Multivalent ligand-lipid constructs

ABSTRACT

Water dispersible, multivalent ligand-lipid constructs that spontaneously and stably incorporate into membranes are disclosed.

This application is a continuation of U.S. application Ser. No.15/528,732 filed, which is the U.S. national phase of InternationalApplication No. PCT/NZ2015/050197 filed Nov. 23, 2015 which designatedthe U.S. and claims priority to Australian Patent Application Nos.2014904722 filed Nov. 21, 2014, and 2015904654 filed Nov. 11, 2015, theentire contents of each of which are hereby incorporated by reference.

This application contains a Sequence Listing which has been filedelectronically in ASCII format.

TECHNICAL FIELD

The invention relates to water dispersible, multivalent ligand-lipidconstructs that spontaneously and stably incorporate into membranes andthe use of such constructs in diagnostic, prognostic, prophylactic andtherapeutic applications. In particular, the invention relates to theuse of the multivalent ligand-lipid constructs in the preparation ofkodecytes with increased avidity for ligand binding proteins.

BACKGROUND ART

The publication of Bovin et al (2005) discloses synthetic molecules thatspontaneously and stably incorporate into lipid bilayers, including cellmembranes. The synthetic molecules consist of a functional moiety (F),such as a mono-, di-, tri- or oligosaccharide, covalently linked to alipid moiety (L), such as phosphatidylethanolamine, via a spacer (S).The spacer is selected to provide synthetic molecules that readilydisperse in water without the use of detergents or solvents and may beused to effect qualitative and quantitative changes in the expression ofcell surface antigens.

The publication discloses the use of these synthetic molecules in amethod of preparing red blood cells expressing controlled amounts ofblood group-related glycans. These modified or transformed cells (nowreferred to as ‘kodecytes’) may be used as positive controls in thequality assurance of blood group typing reagents.

The publication of Bovin et al (2009) discloses functional lipidconstructs consisting of a functional moiety (F) covalently linked to alipid (L) moiety via an elongate spacer (S). In common with thesynthetic molecules disclosed in the publication of Bovin et al (2005),the constructs spontaneously incorporate into cell membranes despitebeing readily dispersible in water. The constructs provide theadditional advantage that the functional moiety (F) is presented at adistance from the surface of the cell membrane. The publication of Bovinet al (2010) discloses constructs where the functional moiety (F) is aligand for a receptor. The publication discloses multiligand constructsof a tri- or tetra-antennary structure. The inter-ligand spacing of theconstructs is intended to promote multivalent interactions between theligands and the ligand-binding protein or receptor.

Ligand binding proteins include glycan binding proteins (GBPs). Theseproteins play important roles in mechanisms of immunity and microbe-hostinteractions. GBPs are present in the sera of all individuals. Theimmune system depends largely on the presence of a competent andwell-equipped repertoire of these GBPs. Many of the GBPs are naturalantibodies (NAbs) that bind to glycan ligands expressed in normal humantissues (auto-antibodies). However, NAbs may also be associated with anumber of diseases, e.g. the antibodies to tumour-associatedcarbohydrate antigens (TACA). Transformation of cells from healthy topre-malignant and malignant is associated with the appearance ofabnormal glycosylation on proteins and lipids presented on the surfaceof the cells. Changes in the NAb profile of an individual can thereforebe associated with the onset and progress of a number of diseases,including cancer.

It is an object of the present invention to provide multivalentligand-lipid constructs for use in the preparation of kodecytes withincreased avidity for ligand binding proteins. The preceding object isto be read in the alternative with the object at least to provide auseful choice.

DISCLOSURE OF INVENTION

In a first aspect the invention provides a multivalent ligand-lipidconstruct of the structure:

where F is a ligand, S is a tetraantennary spacer, and L is a conjugatedphosphatidylethanolamide.

Preferably, S is a tetraantennary spacer of the structure:

where m is the integer 1, 2 or 3 and R is of the structure:

where M is a monovalent cation or substituent, n is the integer 2, 3, 4,5, 6 or 7, and * is the point of attachment of F or L. Preferably, M isH⁺ and n is the integer 5.

Preferably, L is a conjugated phosphatidylethanolamide of the structure:

where M′ is a monovalent cation, p is the integer 3, 4 or 5, W¹ and W²are independently selected from C₁₆₋₂₀-alkyl or mono- or di-unsaturatedC₁₆₋₂₀-alkenyl groups and * is the point of attachment of S.

Preferably, the multivalent ligand-lipid construct comprises the partialstructure:

More preferably, the multivalent ligand-lipid construct comprises thepartial structure:

In a first embodiment of the first aspect of the invention F is anaminoalkylglycoside and the multivalent ligand-lipid construct is of thestructure:

where Glyc is a glycan and q and r are integers independently selectedfrom 1, 2, 3 and 4.

Preferably, Glyc is a glycan selected from the group consisting of:(Neu5Acα6Galβ4GlcNAcβ2Manα)₂3,6Manβ4GlcNAcβ4GlcNAcβ (YDS); Fucα2Galβ(H_(di)); Fucα2Galβ3(Fucα4)GlcNAcβ (Le^(b)); Fucα2Galβ3GlcNAcβ3Galβ4Glcβ(LNFP I); Fucα2Galβ4(Fucα3)GlcNAcβ (Le^(y)); Fucα2Galβ4GlcNAcβ (H2);Galα; Galβ1-3(Fucα1-3)GlcNAc; Galβ1-3(Fucα1-4)GlcNAcβ1-4GlcNAc;Galβ1-3GlcNAcβ1-4GlcNAc; Galβ1-3GlcNAc;Galβ1-4(Fucα1-3)GlcNAcβ1-4GlcNAc; Galβ1-4(Fucα1-3)GlcNAc;Galβ1-4GlcNAcβ1-4GlcNAc; Galβ1-4GlcNAc; Galα3(Fucα2)Galβ (B_(tri));Galα3(Fucα2)Galβ3(Fucα4)GlcNAcβ (Ble^(b)); Galα3(Fucα2)Galβ3GalNAcα(B3); Galα3(Fucα2)Galβ3GalNAcβ (B4); Galα3(Fucα2)Galβ3GlcNAcβ (B1);Galα3(Fucα2)Galβ4(Fucα3)GlcNAcβ (Blew); Galα3(Fucα2)Galβ4GlcNAcβ (B2);Galα3Galβ4GlcNAcβ (Galili); Galα4Galβ4GlcNAcβ (P1); Galα4Galβ4Glcβ (Gb3(P^(k))); Galα4GlcNAcβ (α-LN); GalNAcα3(Fucα2)Galβ (A_(tri));GalNAcα3(Fucα2)Galβ3(Fucα4)GlcNAcβ (ALe^(b));GalNAcα3(Fucα2)Galβ3GalNAcα (A3); GalNAcα3(Fucα2)Galβ3GalNAcβ (A4);GalNAcα3(Fucα2)Galβ3GlcNAcβ (A1); GalNAcα3(Fucα2)Galβ4(Fucα3)GlcNAcβ(ALe^(y)); GalNAcα3(Fucα2)Galβ4GlcNAcβ (A2); GalNAcα3GalNAcβ (Fs2);GalNAcα3GalNAcβ3Galα4Galβ4Glcβ (Fs5); GalNAcα3Galβ (A_(di));GalNAcα3Galβ4GlcNAcβ; GalNAcβ; GalNAcβ3Galα4Galβ4Glcβ (P);GalNH₂α3(Fucα2)Galβ (AcqB); Galβ; Galβ3(Fucα4)GlcNAcβ (Le^(a));Galβ3GalNAcα (TF); Galβ3GalNAcβ4Galβ4Glcβ (GA1); Galβ4(Fucα3)GlcNAcβ(Le^(x)); Galβ4GlcNAcβ3Galβ4GlcNAcβ (i(LN₂)); Galβ4GlcNAcβ3Galβ4Glcβ(LNnT); Galβ4Glcβ (Lac); GlcAβ3[GlcNAcβ4GlcAβ3]_(n)GlcNAc-aminoalditol(hyaluronate); Manα6(Manα3)Manβ (Man₃); Neu5Acα3Galβ4GlcNAcβ(Neu5Ac3′LN); Neu5Acα3Galβ4Glcβ (Neu5Ac3′Lac); Neu5Acα6GalNAcαβ (SiaTn);Neu5Acα6Galβ4GlcNAcβ (Neu5Ac6′LN); Neu5Gcα3Galβ4GlcNAcβ (Neu5Gc3′LN);SAa2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAβ2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAa2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAa2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc;SAa2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc;SAa2-3Galβ1-3(Fucα1-4)GlcNAcβ1-4Gal;SAa2-3Galβ1-3(Fucα1-4)GlcNAcβ1-4GlcNAc; SAa2-3Galβ1-3(Fucα1-4)GlcNAc;SAa2-3Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAa2-3Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAa2-3Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAc;SAa2-3Galβ1-3GlcNAcβ1-4GlcNAc; SAa2-3Galβ1-3GlcNAc;SAa2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)Glc;SAa2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc;SAa2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAa2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAa2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAa2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAa2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAa2-3Galβ1-4(Fucα1-3)GlcNAcβ1-4Gal;SAa2-3Galβ1-4(Fucα1-3)GlcNAcβ1-4GlcNAc; SAa2-3Galβ1-4(Fucα1-3)GlcNAc;SAa2-3Galβ1-4Glc;SAa2-3Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAa2-3Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc;SAa2-3Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAa2-3Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAa2-3Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc;SAa2-3Galβ1-4GlcNAcβ1-4GlcNAc; SAa2-3Galβ1-4GlcNAc;SAa2-6Galβ1-3(Fucα1-4(GlcNAc;SAa2-6Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAa2-6Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAa2-6Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAa2-6Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc;SAa2-6Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc;SAa2-6Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAa2-6Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAa2-6Galβ1-3GlcNAcβ1-4Galβ1-4(Fucα1-3)GlcNAc;SAa2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)Glc;SAa2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc;SAa2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAa2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAa2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAa2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAa2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc; SAa2-6Galβ1-4Glc;SAa2-6Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAa2-6Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc;SAa2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc;SAa2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc;SAa2-6Galβ1-4GlcNAcβ1-3Galβ1-4(Fucα1-3)GlcNAc;SAa2-6Galβ1-4GlcNAcβ1-4GlcNAc; SAa2-6Galβ1-4GlcNAc;SAa2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAc;SAa2-3Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAc;SAa2-6Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAc andSAa2-6Galβ1-4GlcNAcβ1-3Galβ1-3(Fucα1-4)GlcNAc. More preferably, Glyc isa glycan selected from the group consisting of:(Neu5Acα6Galβ4GlcNAcβ2Manα)₂3,6Manβ4GlcNAcβ4GlcNAcβ (YDS); Fucα2Galβ(H_(di)); Fucα2Galβ3(Fucα4)GlcNAcβ (Le^(b)); Fucα2Galβ3GlcNAcβ3Galβ4Glcβ(LNFP I); Fucα2Galβ4(Fucα3)GlcNAcβ (Le^(y)); Fucα2Galβ4GlcNAcβ (H2);Galα; Galα3(Fucα2)Galβ (B_(tri)); Galα3(Fucα2)Galβ3(Fucα4)GlcNAcβ(Ble^(b)); Galα3(Fucα2)Galβ3GalNAcα (B3); Galα3(Fucα2)Galβ3GalNAcβ (B4);Galα3(Fucα2)Galβ3GlcNAcβ (B1); Galα3(Fucα2)Galβ4(Fucα3)GlcNAcβ (Blew);Galα3(Fucα2)Galβ4GlcNAcβ (B2); Galα3Galβ4GlcNAcβ (Galili);Galα4Galβ4GlcNAcβ (P1); Galα4Galβ4Glcβ (Gb3 (P^(k))); Galα4GlcNAcβ(α-LN); GalNAcα3(Fucα2)Galβ (A_(tri));GalNAcα3(Fucα2)Galβ3(Fucα4)GlcNAcβ (ALe^(b));GalNAcα3(Fucα2)Galβ3GalNAcα (A3); GalNAcα3(Fucα2)Galβ3GalNAcβ (A4);GalNAcα3(Fucα2)Galβ3GlcNAcβ (A1); GalNAcα3(Fucα2)Galβ4(Fucα3)GlcNAcβ(ALe^(y)); GalNAcα3(Fucα2)Galβ4GlcNAcβ (A2); GalNAcα3GalNAcβ (Fs2);GalNAcα3GalNAcβ3Galα4Galβ4Glcβ (Fs5); GalNAcα3Galβ (Ac);GalNAcα3Galβ4GlcNAcβ; GalNAcβ; GalNAcβ3Galα4Galβ4Glcβ (P);GalNH₂α3(Fucα2)Galβ (AcqB); Galβ; Galβ3(Fucα4)GlcNAcβ (Le^(a));Galβ3GalNAcα (TF); Galβ3GalNAcβ4Galβ4Glcβ (GA1); Galβ4(Fucα3)GlcNAcβ(Le^(x)); Galβ4GlcNAcβ3Galβ4GlcNAcβ (i(LN₂)); Galβ4GlcNAcβ3Galβ4Glcβ(LNnT); Galβ4Glcβ (Lac); GlcAβ3[GlcNAcβ4GlcAβ3]_(n)GlcNAc-aminoalditol(hyaluronate); Manα6(Manα3)Manβ (Man₃); Neu5Acα3Galβ4GlcNAcβ(Neu5Ac3′LN); Neu5Acα3Galβ4Glcβ (Neu5Ac3′Lac); Neu5Acα6GalNAcαβ (SiaTn);Neu5Acα6Galβ4GlcNAcβ (Neu5Ac6′LN) and Neu5Gcα3Galβ4GlcNAcβ (Neu5Gc3′LN).Most preferably, Glyc is a glycan selected from the group consisting of:Galα3Galβ4GlcNAcβ (Galili) and GalNAcα3Galβ4GlcNAcβ.

In a second embodiment of the first aspect of the invention F is anoligopeptide comprising an N-maleoyl-β-alanine conjugated Cys residueand the multivalent ligand-lipid construct is of the structure:

where Xaa is an amino acid residue and i and j are either zero orintegers the sum of which is in the range 5 to 30 inclusive. Preferably,i is an integer in the range 5 to 30 inclusive and j is zero. Morepreferably, i is the integer 13 and j is zero. Most preferably, theoligopeptide is the peptide of SEQ ID NO: 01.

In a second aspect the invention provides an improved method ofdetecting the presence of a ligand binding protein in a biologicalsample obtained from a subject comprising the steps of:

-   -   contacting the biological sample with a first suspension of        cells modified by incorporation into the membranes of the cells        multivalent ligand-lipid constructs of the first aspect of the        invention to provide a second suspension;    -   adding an amount of anti-subject binding protein to the second        suspension and incubating at a temperature and for a time        sufficient to permit agglutination of the cell; and    -   determining the degree of agglutination, where the ligand        binding protein binds to F of the ligand-lipid constructs of the        first aspect of the invention.

The improvement in the improved method is an increase in avidity,sensitivity and/or specificity of the method of detecting the presenceof a ligand binding protein in a biological sample relative to the useof a monovalent ligand-lipid construct.

In a third aspect the invention provides a method of determining theability of a ligand to induce complement mediated cell lysis in theserum of a subject comprising the steps of:

-   -   contacting a sample of serum obtained from the subject with a        suspension of O group red blood cells modified by incorporation        into the membranes of the cells multivalent ligand-lipid        constructs of the first aspect of the invention; and then    -   monitoring the rate of haemolysis, where F is the ligand.

In a fourth aspect the invention provides a method of treating patientswith tumours by intratumoural injection of a composition consistingessentially of a construct of one or more multivalent ligand-lipidconstructs of the first aspect of the invention.

In the description and claims of this specification the followingacronyms, symbols, terms and phrases have the meaning provided:“affinity” means the strength of the interaction between two entities,e.g. between enzyme and substrate or receptor and ligand; “avidity”means the strength of a binding interaction, e.g. the bindinginteraction of antibody with antigen; “biocompatible” means not harmfulor toxic to living tissue; “comprising” means “including”, “containing”or “characterized by” and does not exclude any additional element,ingredient or step; “consisting of” means excluding any element,ingredient or step not specified except for impurities and otherincidentals; “consisting essentially of” means excluding any element,ingredient or step that is a material limitation; “diagnostic” meansconcerned with the diagnosis of illness or other problems; “DOPE” means1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine; “glycan” means amono-, di-, tri- or oligosaccharide; “kodecyte” means a cell modified byincorporation into the cell membrane of a construct; “PBS” denotesphosphate buffered saline; “PCV” or “pcv” denotes packed cell volume;“plasma” means the colourless fluid part of blood or lymph, in whichcorpuscles or fat globules are suspended; “prognostic” means predictingthe likely cause or occurrence of a disease or ailment; “prophylactic”means intended to prevent disease; “RBC” denotes red blood cell;“reaction product” means the product of a reaction prior topurification; “saline” means a solution of one or more salts; “serum”means the amber-coloured, protein-rich liquid which separates out whenblood coagulates; “synthetic” means prepared by chemical synthesis;“therapeutic” means relating to the healing of disease; “waterdispersible” means, in the context of describing the properties ofconstructs, a stable, single phase system is formed at a temperature of25° C. when the construct is contacted with water at a concentration ofat least 100 μg/mL and in the absence of organic solvents or detergents.

Amino acid residues are identified using the symbols provided in Table 3of Appendix 2 of Annex C of the Administrative Instructions under thePatent Corporation Treaty (as in force from 1 Jul. 2015). “Functionallysimilar amino acid” means an amino acid with similar propertiesaccording to the following groupings: neutral-weakly hydrophobic (Ala,Gly, Pro, Ser, Thr); hydrophilic-acid amine (Asn, Asp, Gln, Glu);hydrophilic-basic (Arg, His, Lys); hydrophobic (Ile, Met, Leu, Val);hydrophobic-aromatic (Phe, Trp, Tyr) and cross-linking (Cys).

Saccharide residues and their derivatives are identified using thesymbols provided in Table 2 and the appendix of the publication ofMcNaught (1996). Specifically, the following symbols have the meaningprovided: “Abe” means abequose; “All” means allose; “Alt” means altrose;“Api” means apiose; “Ara” means arabinose; “dRib” means 2-deoxyribose;“Fru” means fructose; “Fuc” means fucose; “Fuc-ol” means fucitol; “Gal”means galactose; “Gal” means galactose; “GalN” means galactosamine;“GalNAc” means N-acetylgalactosamine; “Glc” means glucose; “GlcA” meansglucuronic acid; “GlcN” means glucosamine; “GlcN3N” means2,3-diamino-2,3-dideoxy-D-glucose; “GlcNAc” means N-acetylglucosamine;“Glc-ol” means glucitol; “GlcpA6Et” means ethyl glucopryanuronate; “Gul”means gulose; “Gul” means gulose; “Ido” means idose; “IdoA” meansiduronic acid; “Kdo” means 3-deoxy-D-manno-oct-2-ulosonic acid; “Lyx”means lyxose; “Man” means mannose; “Mur” means muramic acid; “Neu” meansneuraminic acid; “Neu2en5Ac” means N-acetyl-2-deoxyneur-2-enaminic acid;“Neu5Ac” means n-acetylneuraminic acid; “Neu5Gc” meansN-glucoloylneuraminic acid; “Psi” means psicose; “Qui” means quinovose;“Rha” means rhamnose; “Rha3,4Me₂” means 3,4-di-O-methylrhamnose; “Rib”means ribose; “Rib5P” means ribose 5-phosphate; “Ribulo (or Rul)” meansribulose; “SA” means sialic acid; Sor” means sorbose; “Tag” meanstagatose; “Tal” means talose; “Xyl” means xylose; “Xyl2CMe” means2-C-methylxylose; “Xylulo (or Xul)” means xylulose and “β-D-Galp4S”means β-D-galactopyranose 4-sulfate.

The terms “first”, “second”, “third”, etc. used with reference toelements, features or integers of the subject matter defined in theStatement of Invention and Claims, or when used with reference toalternative aspects or embodiments of the invention are not intended toimply an order of preference.

Where concentrations or ratios of reagents are specified theconcentration or ratio specified is the initial concentration or ratioof the reagents. Where values are expressed to one or more decimalplaces standard rounding applies. For example, 1.7 encompasses the range1.650 recurring to 1.749 recurring.

In the absence of further limitation the use of plain bonds in therepresentations of the structures of compounds encompasses thediastereomers, enantiomers and mixtures thereof of the compounds. In therepresentations of the structures, partial structures or substructuresof constructs the repeat of a divalent radical is represented by:

where —X— is the divalent radical repeated n times. Where the divalentradical is methylene (—CH₂—) the repeat of this divalent radical isrepresented by:

The invention will now be described with reference to embodiments orexamples and the figures of the accompanying drawings pages.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Alternative representation of the construct designated(MUT21-Mal-βAla-CMG3-NHCH₂)₃CCH₂NH-CMG3-Ad-DOPE (26).

FIG. 2. Photograph of test tubes following complement induced lysis. Thenotations in the photograph correspond to the use of the followingconstructs in the preparation of the kodecytes (at the concentrationsindicated): Gal SA1-L1 (49), Gal T17 (35), Gal CMG 2 (47) and GalNAcalpha1 (48).

DESCRIPTION OF EMBODIMENTS

The multivalent presentation of ligands is particularly advantageouswhere the ligands are glycans. The affinities of glycan-binding proteins(GBPs) for glycan ligands in the monovalent state are generally verylow. The multivalent presentation of glycan ligands permits GBPs such asantibodies to bind with increased avidity. In general, the multivalentpresentation of glycan ligands amplifies differences in specificity ofbinding of GBPs relative to the low intrinsic affinities of GBPs fortheir glycan ligands. As a result the presence of GBPs in human sera maybe detected using simple agglutination or cell lysis assays.

Chemistry

Preparation of (Boc-Gly₂-HNCH₂)₄C (3) (Step i of Scheme I)

Tetraamine (H₂N—CH₂)₄C (1) was synthesized according the methoddisclosed in the publication of Litherland et al (1938). To a stirredsolution of the tetraamine 1 (500 mg, 1.52 mmol) in a mixture of 1Maqueous NaHCO₃ (18.2 ml) and i-PrOH (9 ml), Boc-GlyGlyNos (2) (4012 mg,12.18 mmol) was added (CO₂ evolution, foaming). The reaction mixture wasstirred for 30 min, then 6 ml of 1M aqueous NaHCO₃ was added and themixture stirred overnight. Precipitate of (Boc-Gly₂-HNCH₂)₄C (3) wasfiltered, washed thoroughly with methanol/water mixture (1:1, 20 ml) anddried in vacuum. Yield 1470 mg (98%), white solid.

¹H NMR (500 MHz, [D₆]DMSO, 30° C.) δ, ppm: 8.491 (t, J=5.6 Hz, 1H;NHCO), 7.784 (t, J=6.6 Hz, 1H; C—CH₂—NHCO), 6.858 (t, J=6 Hz, 1H;NHCOO), 3.696 (d, J=5.6 Hz, 2H; COCH ₂NH), 3.675 (d, J=6 Hz, 2H; COCH₂NHCOO), 2.685 (d, J=6.6 Hz, 2H; C—CH ₂NH), 1.375 (s, 9H; C(CH₃)₃.

Preparation of (CF₃COOH*H-Gly₂-NHCH₂)₄C (4) (Step ii of Scheme I)

The (Boc-Gly₂-HNCH₂)₄C (3) (1450 mg, 1.466 mmol) was dissolved inCF₃COOH (5 ml) and the solution was kept for 2 h at room temperature.Trifluoroacetic acid was removed under vacuum and the residue was threetimes extracted with (CH₃CH₂)₂O (slight agitation with 30 ml of(CH₃CH₂)₂O for 30 min., followed by decantation) to eliminate residualCF₃COOH. Solid residue was dried under vacuum, dissolved in a minimumvolume of water and passed through a Sephadex LH-20 column and elutdwith water. Fractions, containing product 4, were combined, evaporatedto c. 5 ml and freeze dried. Yield 1424 mg (93%), white solid. TLC:R_(f) 0.5 (ethanol/conc. NH₃; 2:1 (v/v)).

¹H NMR (500 MHz, [D₂]H₂O, 30° C.) δ, ppm: 4.028 (s, 2H; COCH ₂NH), 3.972(s, 2H; COCH ₂NH), 2.960 (s, 2H; C—CH ₂NH).

Preparation of([2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxy-carbonylmethyl-amino)-aceticacid methyl ester (7) (Step i of Scheme II)

To a stirred solution of (methoxycarbonylmethyl-amino)-acetic acidmethyl ester hydrochloride (5) (988 mg, 5 mmol) in DMF (15 ml)Boc-GlyGlyNos (2) (3293 mg, 10 mmol) and (CH₃CH₂)₃N (3475 μL, 25 mmol)were added. The mixture was stirred overnight at room temperature andthen diluted with o-xylene (70 ml) and evaporated. Flash columnchromatography on silica gel (packed in toluene, and eluted with ethylacetate) resulted in a crude product. The crude product was dissolved inchloroform and washed sequentially with water, 0.5 M NaHCO₃ andsaturated KCl. The chloroform extract was evaporated and the productpurified on a silica gel column (packed in chloroform and eluted with15:1 (v/v) chloroform/methanol). Evaporation of the fractions and dryingunder vacuum of the residue provided a colourless thick syrup of product7. Yield 1785 mg, (95%). TLC: R₁=0.49 (7:1 (v/v) chloroform/methanol).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.) δ, ppm: 7.826 (t, J=5.1 Hz, 1H;NHCO), 6.979 (t, J=5.9 Hz, 1H; NHCOO), 4.348 and 4.095 (s, 2H; NCH₂COO), 3.969 (d, J=5.1 Hz, 2H; COCH ₂NH), 3.689 and 3.621 (s, 3H; OCH₃), 3.559 (d, J=5.9 Hz, 2H; COCH ₂NHCOO), 1.380 (s, 9H; C(CH₃)₃).

Preparation of{[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-aceticAcid (8) (Step ii of Scheme II)

To a stirred solution of 7 (1760 mg, 4.69 mmol) in methanol (25 ml) 0.2M aqueous NaOH (23.5 ml) was added and the solution kept for 5 min atroom temperature. The solution was then acidified with acetic acid (0.6ml) and evaporated to dryness. Column chromatography of the residue onsilica gel (packed in ethyl acetate and eluted with 2:3:1 (v/v/v)i-PrOH/ethyl acetate/water) resulted in a recovered 7 (63 mg, 3.4%) andtarget compound 8 (1320 mg). The intermediate product was then dissolvedin methanol/water/pyridine mixture (20:10:1, 30 ml) and passed throughan ion exchange column (Dowex 50X4-400, pyridine form, 5 ml) to removeresidual sodium cations. The column was then washed with the samesolvent mixture, the eluant evaporated, the residue dissolved inchloroform/benzene mixture (1:1, 50 ml) and then evaporated and driedunder vacuum. Yield of product 8 was 1250 mg (74%), white solid. TLC:R_(f) 0.47 (4:3:1 (v/v/v) i-PrOH/ethyl acetate/water).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.), mixture of cis- and trans-conformersof N-carboxymethylglycine unit c.3:1. Major conformer; δ, ppm: 7.717 (t,J=5 Hz, 1H; NHCO), 7.024 (t, J=5.9 Hz, 1H; NHCOO), 4.051 (s, 2H; NCH₂COOCH₃), 3.928 (d, J=5 Hz, 2H; COCH ₂NH), 3.786 (s, 2H; NCH ₂COOH),3.616 (s, 3H; OCH ₃), 3.563 (d, J=5.9 Hz, 2H; COCH ₂NHCOO), 1.381 (s,9H; C(CH₃)₃) ppm; minor conformer, 8=7.766 (t, J=5 Hz, 1H; NHCO), 7.015(t, J=5.9 Hz, 1H; NHCOO), 4.288 (s, 2H; NCH ₂COOCH₃), 3.928 (d, J=5 Hz,2H; COCH ₂NH), 3.858 (s, 2H; NCH ₂COOH), 3.676 (s, 3H; OCH ₃), 3.563 (d,J=5.9 Hz, 2H; COCH ₂NHCOO), 1.381 (s, 9H; C(CH₃) 3).

Preparation of([2-(2-tert-Butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino)-aceticacid N-oxysuccinimide ester (Boc-Gly₂(MCMGly)Nos) (9) (Step iii ofScheme III)

To an ice-cooled stirred solution of 8 (1200 mg, 3.32 mmol) andN-hydroxysuccinimide (420 mg, 3.65 mmol) in DMF (10 ml) was addedN,N′-dicyclohexylcarbodiimide (754 mg, 3.65 mmol). The mixture wasstirred at 0° C. for 30 min, then for 2 hours at room temperature. Theprecipitate of N,N′-dicyclohexylurea was filtered off, washed with DMF(5 ml), and filtrates evaporated to a minimal volume. The residue wasthen agitated with (CH₃CH₂)₂O (50 ml) for 1 hour and an ether extractremoved by decantation. The residue was dried under vacuum providing theester 9 (1400 mg, 92%) as a white foam. TLC: R_(f) 0.71 (40:1 (v/v)acetone/acetic acid).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.), mixture of cis- and trans-conformersof N-carboxymethylglycine unit c. 3:2.

Major conformer; δ, ppm: 7.896 (t, J=5.1 Hz, 1H; NHCO), 6.972 (t, J=5.9Hz, 1H; NHCOO), 4.533 (s, 2H; NCH ₂COON), 4.399 (s, 2H; NCH ₂COOCH₃),3.997 (d, J=5.1 Hz, 2H; COCH ₂NH), 3.695 (s, 3H; OCH ₃), 3.566 (d, J=5.9Hz, 2H; COCH ₂NHCOO), 1.380 (s, 9H; C(CH₃)₃).

Minor conformer; δ, ppm: 7.882 (t, J=5.1 Hz, 1H; NHCO), 6.963 (t, J=5.9Hz, 1H; NHCOO), 4.924 (s, 2H; NCH ₂COON), 4.133 (s, 2H; NCH ₂COOCH₃),4.034 (d, J=5.1 Hz, 2H; COCH ₂NH), 3.632 (s, 3H; OCH ₃), 3.572 (d, J=5.9Hz, 2H; COCH ₂NHCOO), 1.380 (s, 9H; C(CH₃)₃).

The ester 9 (1380 mg) was dissolved in DMSO to provide a volume of 6 mland used as a 0.5 M solution (stored at −18° C.).

Preparation of {Boc-[Gly₂(MCMGly)]Gly₂-NHCH₂}₄C (10) (Step i of SchemeIII)

To a stirred solution of (CF₃COOH.H-Gly₂-HNCH₂)₄C (4) (277 mg, 0.265mmol) in DMSO (2 ml) the ester 9 (1.591 mmol, 3.18 ml of 0.5 M solutionin DMSO) and (CH₃CH₂)₃N (295 μL, 2.121 mmol) were added. The mixture wasstirred overnight at room temperature, acidified with 150 μL AcOH andsolvent removed under vacuum (freeze drying). The residue was extractedthree times with (CH₃CH₂)₂O (slight agitation with 20 ml of (CH₃CH₂)₂Ofor 30 min followed by decantation). The solid residue was dissolved ina minimal volume of acetone and fractionated on silica gel column(packed in acetone and eluted with acetone, 20:2:1 (v/v/v)acetone/methanol/water and 15:2:1 (v/v/v) acetone/methanol/water).Selected fractions were evaporated and the residue was dried undervacuum. The yield of pure {Boc-[Gly₂(MCMGly)]Gly₂-NHCH₂}₄C (10) was 351mg (68%), white solid. TLC: R_(f) 0.38 (15:2:1 (v/v/v)acetone/methanol/water).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.), mixture of cis- and trans-conformersof N-carboxymethylglycine unit in chain c. 3:2.

Major conformer; δ, ppm: 8.593 (t, J=5 Hz, 1H; NHCO), 8.335 (t, J=5.4Hz, 1H; NHCO), 7.821 (t, J=6.4 Hz, 1H; C—CH₂—NHCO), 7.786 (t, J=5.1 Hz,1H; NHCO), 6.993 (t, J=6 Hz, 1H; NHCOO), 4.139 (s, 2H; NCH ₂CO), 4.074(s, 2H; NCH ₂COO(CH₃)), 3.985 (d, J=5 Hz, 2H; COCH ₂NH), 3.887 (d, J=5.4Hz, 2H; COCH ₂NH), 3.726 (d, J=5.1 Hz, 2H; COCH ₂NH), 3.634 (s, 3H; OCH₃), 3.567 (d, J=6 Hz, 2H; COCH ₂NHCOO), 2.686 (broad. d, J=6.4 Hz, 2H;C—CH ₂NH), 1.379 (s, 9H; C(CH₃)₃).

Minor conformer; δ, ppm: 8.511 (t, J=5 Hz, 1H; NHCO), 8.158 (t, J=5.4Hz, 1H; NHCO), 7.821 (t, J=6.4 Hz, 1H; C—CH₂—NHCO), 7.786 (t, J=5.1 Hz,1H; NHCO), 6.993 (t, J=6 Hz, 1H; NHCOO), 4.292 (s, 2H; NCH ₂CO), 3.998(s, 2H; NCH ₂COOCH₃), 3.954 (d, J=5 Hz, 2H; COCH ₂NH), 3.826 (d, J=5.4Hz, 2H; COCH ₂NH), 3.715 (d, J=5.1 Hz, 2H; COCH ₂NH), 3.692 (s, 3H; OCH₃), 3.567 (d, J=6 Hz, 2H; COCH ₂NHCOO), 2.686 (broad. d, J=6.4 Hz, 2H;C—CH ₂NH), 1.379 (s, 9H; C(CH₃)₃).

Preparation of (CF₃COOH*H-[Gly₂ (MCMGly)]Gly₂-NHCH₂)₄C (11) (Step ii ofScheme III)

The {Boc-[Gly₂(MCMGly)]Gly₂-NHCH₂}₄C (10) (330 mg, 0.168 mmol) wasdissolved in CF₃COOH (2 ml) and the solution was kept for 40 min at roomtemperature. Trifluoroacetic acid was evaporated under vacuum, theresidue extracted three times with (CH₃CH₂)₂O (slight agitation with 20ml of (CH₃CH₂)₂O for 30 min followed by decantation) to eliminateresidual CF₃COOH, and then dried under vacuum. The yield of{CF₃COOH.H-[Gly₂(MCMGly)]Gly₂-NHCH₂}₄C (11) was 337 mg (99%), whitesolid.

¹H NMR (500 MHz, [D₂]H₂O, 30° C.), mixture of cis- and trans-conformersof N-carboxymethylglycine unit in chain c. 11:10.

Major conformer; δ, ppm: 4.370 (s, 2H; NCH ₂CO), 4.265 (s, 2H; NCH₂COOCH₃), 4.215 (s, 2H; COCH ₂NH), 4.138 (s, 2H; COCH ₂NH), 3.968 (s,2H; COCH ₂NH), 3.919 (s, 2H; COCH ₂NH₂), 3.775 (s, 3H; OCH ₃), 2.914 (s,2H; C—CH ₂NH).

Minor conformer; δ, ppm: 4.431 (s, 2H; NCH ₂CO), 4.241 (s, 2H; NCH₂COOCH₃), 4.239 (s, 2H; COCH ₂NH), 4.074 (s, 2H; COCH ₂NH), 3.960 (s,2H; COCH ₂NH), 3.919 (s, 2H; COCH ₂NH₂), 3.829 (s, 3H; OCH ₃), 2.914 (s,2H; C—CH ₂NH).

Preparation of (CF₃COOH.H-[Gly₂(MCMGly)]₂Gly₂-NHCH₂)₄C (13) (Steps i andii of Scheme IV)

To a stirred solution of (CF₃COOH.H-[Gly₂ (MCMGly)]Gly₂-HNCH₂)₄C (11)(272 mg, 0.135 mmol) in DMSO (2 ml) the ester 9 (0.809 mmol, 1.62 ml of0.5 M solution in DMSO) and (CH₃CH₂)₃N (112 μL, 0.809 mmol) were added.The mixture was stirred overnight at room temperature, acidified with 70μL AcOH and solvent removed under vacuum (freeze drying). The residuewas extracted three times with (CH₃CH₂)₂O (slight agitation with 15 mlof (CH₃CH₂)₂O for 30 min followed by decantation). Solid residue wasdissolved in a minimal volume of 7:1 (v/v) acetone/methanol mixture andfractionated on a silica gel column (packed in acetone and eluted with7:1 (v/v) acetone/methanol, 10:2:1 (v/v/v), 9:2:1 (v/v/v), 8:2:1 (v/v/v)acetone/methanol/water). Selected fractions were evaporated and theresidue was dried in vacuum. The yield of pure{Boc-[Gly₂(MCMGly)]₂Gly₂-NHCH₂}₄C (12) was 279 mg (71%), white solid.TLC: R_(f) 0.42 (8:2:1 (v/v/v) acetone/methanol/water).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.), mixture of conformers by twoN-carboxymethyl-glycine units per chain, δ, ppm: 8.604, 8.519, 8.397,8.388, 8.346, 8.211, 8.200, 8.167, 8.034, 8.024, 7.925, 7.912, 7.819 and7.773 (t, 6H; 6NHCO), 6.992 (t, J=5.9 Hz, 1H; NHCOO), 4.302-3.723 (18H;2NCH ₂CO, 2NCH ₂COOCH₃, 5COCH ₂NH), 3.692, 3.689 and 3.632 (s, 6H; 2OCH₃), 3.566 (d, J=5.9 Hz, 2H; COCH ₂NHCOO), 2.686 (broad. d, 2H; C—CH₂NH), 1.380 (s, 9H; C(CH₃)₃).

The {Boc-[Gly₂(MCMGly)]₂Gly₂-NHCH₂}₄C (12) (269 mg, 91.65 μmol) wasdissolved in CF₃COOH (2 ml) and the solution was kept for 40 min at roomtemperature. Trifluoroacetic acid was evaporated under vacuum, theresidue extracted three times with (CH₃CH₂)₂O (slight agitation with 15ml of (CH₃CH₂)₂O for 30 min followed by decantation) to remove residualCF₃COOH, and then dried under vacuum. The yield of{CF₃COOH.H-[Gly₂(MCMGly)]₂Gly₂-NHCH₂}₄C (13) was 270 mg (98%), whitesolid.

¹H NMR (500 MHz, [D₂]H₂O, 30° C.), mixture of conformers by twoN-carboxymethyl-glycine units per chain, δ, ppm: 4.441-3.963 (singlets,18H; 2 NCH ₂CO, 2NCH ₂COOCH₃, 5 COCH2NH), 3.920 (s, 2H; COCH ₂NH₂ ⁺,3.833, 3.824, 3.780 and 3.773 (s, 6H; 2OCH ₃), 2.918 (s, 2H; C—CH ₂NH).

Preparation of {CF₃COOH.H-[Gly₂(MCMGly)]₃Gly-NHCH₂}₄C (15) (Steps iiiand iv of Scheme IV)

To a stirred solution of (CF₃COOH H-[Gly₂(MCMGly)]₂Gly₂-HNCH₂)₄C (13)(175 mg, 58.5 μmol) in DMSO (2 ml) the ester 9 (0.351 mmol, 0.702 ml of0.5 M solution in DMSO) and (CH₃CH₂)₃N (49 μL, 0.351 mmol) were added.The mixture was stirred overnight at room temperature, acidified with 30μL AcOH and solvent removed under vacuum (freeze drying). The residuewas dissolved in a minimal volume of a mixture of 1:1 (v/v)acetonitrile/water and fractionated on a Sephadex LH-20 column (elutedwith 1:1 (v/v) acetonitrile/water). Selected fractions were evaporatedand the residue was dried in vacuum. The yield of pure {Boc-[Gly₂(MCMGly)]₃Gly₂-NHCH₂}₄C (14) was 279 mg (71%), white solid. TLC: R_(f)0.42 (8:2:1 (v/v/v) acetone/methanol/water). Fractions containing{Boc-[Gly₂(MCMGly)]₃Gly₂-NHCH₂}₄C (14) were combined, evaporated to c. 2ml volume and freeze dried. The initial yield was 215 mg (94%).Additional purification on a silica gel column (packed in acetonitrileand eluted with 4:5:2 (v/v/v) i-PrOH/acetonitrile/water) resulted in 169mg of Boc-[Gly₂(MCMGly)]₃Gly₂-NHCH₂)₄C (yield 74%, white solid). TLC:R_(f) 0.45 (4:5:2 (v/v/v) i-PrOH/acetonitrile/water).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.), mixture of conformers by threeN-carboxymethyl-glycine units per chain, δ, ppm: 8.594-7.772 (triplets,together 8H; 8NHCO), 6.989 (t, J=5.6 Hz, 1H; NHCOO), 4.303-3.722 (26H;3NCH ₂CO, 3NCH ₂COOCH₃, 7COCH ₂NH), 3.692 and 3.632 (s, 9H; 3OCH ₃),3.565 (d, J=5.6 Hz, 2H; COCH ₂NHCOO), 2.687 (broad. d, 2H; C—CH ₂NH),1.380 (s, 9H; C(CH₃)₃).

The {Boc-[Gly₂(MCMGly)]₃Gly₂-NHCH₂}₄C (146 mg, 37.36 μmol) (14) wasdissolved in CF₃COOH (1 ml) and the solution was kept for 40 min at roomtemperature. Trifluoroacetic acid was evaporated under vacuum, theresidue extracted three times with (CH₃CH₂)₂O (slight agitation with 10ml of (CH₃CH₂)₂O for 30 min followed by decantation) to remove residualCF₃COOH, and then dried under vacuum. The yield of{CF₃COOH.H-[Gly₂(MCMGly)]₃Gly₂-NHCH₂}₄C (15) was 147 mg (99%), whitesolid.

¹H NMR (500 MHz, [D₂]H₂O, 30° C.), mixture of conformers by threeN-carboxymethyl-glycine units per chain, δ, ppm: 4.446-3.964 (singlets,26H; 3NCH ₂CO, 3NCH ₂COOCH₃, 7COCH ₂NH), 3.924 (s, 2H; COCH ₂NH₂),3.836, 3.828, 3.824, 3.783, 3.778 and 3.773 (s, 9H; 3OCH ₃), 2.919 (s,2H; C—CH ₂NH).

Preparation of (CF₃COOH.H-[Gly₂(MCMGly)]₄Gly₂-NHCH₂)₄C (17) (Steps v andvi of Scheme IV)

To a stirred solution of (CF₃COOH.H-Gly₂(MCMGly)]₃-HNCH₂)₄C (15) (68 mg,17.16 μmol) in DMSO (1 ml) the ester 9 (0.137 mmol, 0.275 ml of 0.5 Msolution in DMSO) and (CH₃CH₂)₃N (14.3 μL, 0.103 mmol) were added. Themixture was stirred overnight at room temperature, acidified with 100 μLAcOH and solvent removed under vacuum (freeze drying). The residue wasdissolved in a minimal volume of a mixture of 1:1 (v/v)acetonitrile/water (0.25% AcOH) and fractionated on a Sephadex LH-20column (eluted with 1:1 (v/v) acetonitrile/water (0.25% AcOH)).Fractions containing {Boc-[Gly₂(MCMGly)]₄Gly₂-NHCH₂}₄C (16) werecombined, evaporated to c. 2 ml volume and freeze dried. The yield was81 mg (96%), white solid. TLC: R_(f) 0.24 (4:5:2 (v/v/v)i-PrOH/acetonitrile/water).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.), mixture of conformers by fourN-carboxymethyl-glycine units per chain, δ, ppm: 8.590-7.773 (triplets,10H; 10NHCO), 6.989 (t, J=5.6 Hz, 1H; NHCOO), 4.303-3.722 (34H; 4NCH₂CO, 4NCH ₂COOCH₃, 9COCH ₂NH), 3.691 and 3.631 (s, 12H; 4OCH ₃), 3.565(d, J=5.6 Hz, 2H; COCH ₂NHCOO), 2.684 (broad. d, 2H; C—CH ₂NH), 1.379(s, 9H; C(CH₃)₃).

The {Boc-[Gly₂(MCMGly)]₄Gly₂-NHCH₂}₄C (16) (74 mg, 15.16 μmol) wasdissolved in CF₃COOH (1 ml) and the solution was kept for 40 min at roomtemperature. Trifluoroacetic acid was evaporated under vacuum, theresidue extracted three times with (CH₃CH₂)₂O (slight agitation with 10ml of (CH₃CH₂)₂O for 30 min followed by decantation) to remove residualCF₃COOH, and then dried under vacuum. The yield of{CF₃COOH.H-[Gly₂(MCMGly)]₄Gly₂-NHCH₂}₄C (17) was 72 mg (96%), whitesolid.

¹H NMR (500 MHz, [D₂]H₂O, 30° C.), mixture of conformers by fourN-carboxymethyl-glycine units per chain, δ, ppm: 4.446-3.964 (singlets,34H; 4NCH ₂CO, 4NCH ₂COOCH₃, 9COCH ₂NH), 3.925 (s, 2H; COCH ₂NH₂—),3.836, 3.829, 3.827, 3.822, 3.783, 3.779, 3.777 and 3.772 (s, 12H; 4OCH₃), 2.919 (s, 2H; C—CH ₂NH).

Preparation of (CF₃COOH.H-[Gly₂(MCMGly)]₅Gly₂-NHCH₂)₄C (19) (Steps viiand viii of Scheme IV)

To a stirred solution of (CF₃COOH H-Gly₂ (MCMGly)]₄-HNCH₂)₄C (17) (16.8mg, 3.403 μmol) in DMSO (1 ml) the ester 9 (27.2 μmol, 63 μl of 0.5 Msolution in DMSO) and (CH₃CH₂)₃N (3 μl, 21.6 μmol) were added. Themixture was stirred overnight at room temperature, acidified with 100 μLAcOH and solvent removed under vacuum (freeze drying). The residue wasdissolved in a minimal volume of a mixture of 1:1 (v/v)acetonitrile/water (0.25% AcOH) and fractionated on a Sephadex LH-20column (eluted with 1:1 (v/v) acetonitrile/water (0.25% AcOH)).Fractions containing {Boc-[Gly₂(MCMGly)]Gly₂-NHCH₂}₄C (18) werecombined, evaporated to c. 1 ml volume and freeze dried. The yield was19 mg (95%), white solid. TLC: R_(f) 0.15 (4:3:2 (v/v/v)i-PrOH/acetonitrile/water).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.), mixture of conformers by fiveN-carboxymethyl-glycine units per chain, δ, ppm: 8.595-7.772 (triplets,12H; 12NHCO), 6.989 (t, J=5.6 Hz, 1H; NHCOO), 4.303-3.723 (42H; 5 NCH₂CO, 5NCH ₂COOCH₃, 11COCH ₂NH), 3.692 and 3.631 (s, 15H; 5OCH ₃), 3.565(d, J=5.6 Hz, 2H; COCH ₂NHCOO), 2.686 (broad. d, 2H; C—CH ₂NH), 1.380(s, 9H; C(CH₃)₃).

The {Boc-[Gly₂(MCMGly)]₅Gly₂-NHCH₂}₄C (18) (19 mg, 3.25 μmol) wasdissolved in CF₃COOH (0.5 ml) and the solution was kept for 40 min atroom temperature. Trifluoroacetic acid was evaporated under vacuum, theresidue extracted three times with (CH₃CH₂)₂O (slight agitation with 5ml of (CH₃CH₂)₂O for 30 min followed by decantation) to remove residualCF₃COOH, and then dried under vacuum. Yield of{CF₃COOH.H-[Gly₂(MCMGly)]₅Gly₂-NHCH₂}₄C (19) was 20 mg (99%), whitesolid.

¹H NMR (500 MHz, [D₂]H₂O, 30° C.), mixture of conformers by fiveN-carboxymethyl-glycine units per chain, δ, ppm: 4.446-3.965 (singlets,42H; 5NCH ₂CO, 5NCH ₂COOCH₃, 11COCH ₂NH), 3.924 (s, 2H; COCH ₂NH₂ ⁺),3.835, 3.829, 3.827, 3.825, 3.823, 3.783, 3.779, 3.777 and 3.773 (s,15H; 5OCH ₃), 2.919 (s, 2H; C—CH ₂NH).

Preparation of [CF₃COOH.H-(Gly₂CMGly)₅Gly₂-NHCH]₄C, Et₃N-salt (20)(Scheme IV)

To a solution of product 19 (463 mg, 0.07835 mmol) in water (26 mL),Et₃N (523 μL, 3.761 mmol) was added and the solution kept for 18 h atr.t. After evaporation the residue was freeze-dried in vacuum. Yield ofproduct 20 was 587 mg (98%), white solid. TLC: R_(f) 0.39 (1:2:1 (v/v/v)CHCl₃/MeOH/water).

¹H NMR (600 MHz, [D₂]H₂O, 30° C.) δ, ppm: 4.309-3.919 (176H; 20NCH ₂CO,20NCH ₂COOH, 48COCH ₂NH), 3.226 (q, 120H, J=7.3 Hz; 60NCH ₂CH₃), 2.964(broad.s, 8H; 4C—CH ₂NH), 1.305 (t, 180H, J=7.3 Hz; 60NCH₂CH ₃).

MALDI TOF mass-spectrum, M/Z: 5174, M+H; 5196, M+Na.

Preparation of activated1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (DE-Ad-OSu) (23)(Step i of Scheme V)

To a solution of bis(N-hydroxysuccinimidyl) adipate (21) (70 mg, 205μmol) in dry N,N-dimethylformamide (1.5 ml),1,2-O-dioleoyl-sn-glycero-3-phosphatidylethanolamine (22) (40 μmol) inchloroform (1.5 ml) was added, followed by triethylamine (7 μl). Themixture was kept for 2 h at room temperature, then neutralized withacetic acid and partially concentrated under vacuum. Columnchromatography (Sephadex LH-20, 1:1 chloroform-methanol, 0.2% aceticacid) of the residue yielded the product 23 (37 mg, 95%) as a colorlesssyrup.

¹H NMR (CDCl₃/CD₃OD, 2:1) 5.5 (m, 4H, 2×(—CH═CH—), 5.39 (m, 1H, —OCH₂—CHO—CH₂O—), 4.58 (dd, 1H, J=3.67, J=11.98, —CCOOHCH—CHO—CH₂O—), 4.34(dd, 1H, J=6.61, J=11.98, —CCOOHCH—CHO—CH₂O—), 4.26 (m, 2H, PO—CH₂—CH₂—NH₂), 4.18 (m, 2H, —CH₂ —OP), 3.62 (m, 2H, PO—CH₂—CH₂ —NH₂), 3.00 (s,4H, ONSuc), 2.8 (m, 2H, —CH₂ —CO (Ad), 2.50 (m, 4H, 2×(—CH₂ —CO), 2.42(m, 2H, —CH₂ —CO (Ad), 2.17 (m, 8H, 2×(—CH₂ —CH═CH—CH₂ —), 1.93 (m, 4H,COCH₂CH₂ CH₂ CH₂CO), 1.78 (m, 4H, 2×(COCH₂CH₂ —), 1.43, 1.47 (2 bs, 40H,20 CH₂), 1.04 (m, 6H, 2CH₃). R_(f) 0.5 (chloroform-methanol-water,6:3:0.5.

Preparation of[H-(Gly₂CMGly)₅Gly₂-NHCH₂]₃[DE-CO(CH₂)₄CO-(Gly₂CMGly)₅Gly₂-NHCH₂]C, Na,Et₃N-salt (24) (Step ii of Scheme V)

To a stirred solution of product 20 (522 mg, 0.06821 mmol) inwater/2-propanol mixture (16 mL, 2:3) 1M NaHCO₃ (547 μL, 0.547 mmol) anda solution of DE-Ad-OSu (23) (66.1 mg, 0.06821 mmol) in dichloroethane(368 μL) were added, and the solution was stirred for 1.5 h at r.t.After acidification with AcOH (94 μL) the solution was evaporated andthe residue was dried in vacuum. Dried mixture was dissolved in 3 mL ofwater/MeOH (15:1) and put on a C18 reverse phase column (˜45 mL of phasewashed with 75% MeOH and then with water/MeOH 15:1). Substances wereeluted sequentially with water/MeOH (15:1-50 mL; 9:1-50 mL; 7.5:2.5-50mL; 1:1-50 mL; 2.5:7.5-100 mL). Unreacted 20 was eluted with water/MeOH15:1 (Na salt by NMR data, 116 mg, 30.8% of recovery) and withwater/MeOH 9:1 (Et₃N salt by NMR data, 63 mg, 13.6% of recovery). Target(H-CMG₅)₃C(CMG₅-Ad-DE) (24) was eluted with water/MeOH 1:1. Yield ofpure freeze-dried product 24 was 135 mg (25.5% on (24)), white solid.TLC (1:2:1 (v/v/v) MeOH/ethyl acetate/water): 20 R_(f) 0.06; 24 R_(f)0.17.

(H-CMG₅)₃C(CMG₅-Ad-DE) Na (Et₃N)₂O (24): ¹H NMR (700 MHz,[D₂]H₂O/[D₄]CH₃OH 2:1 (v/v), 30° C.) δ, ppm: 5.561 (m, 4H; 2 cis CH═CHof DE), 5.454 (m, 1H; OCH ₂—CH(OCO)CH₂O of DE), 4.629 (dd, 1H, J=12.3Hz/2 Hz; OCH₂—CH(OCO)CHOCO of DE), 4.462-4.057 (181H; 20NCH ₂CO, 20NCH₂COOH, 48 COCH ₂NH, OCH ₂—CH(OCO)CHOCO of DE, OCH ₂CH₂NH of DE), 3.597(t, 2H, J=5 Hz; OCH₂CH ₂NH of DE), 3.226 (q, 102H, J=7.3 Hz; 51NCH₂CH₃), 3.099 (broad.s, 8H; 4C—CH ₂NH), 2.557, 2.532, 2.522 and 2.456(triplets, total 8H; 4CO—CH ₂CH₂), 2.203 (˜dd, 8H, J=12 Hz/5.8 Hz; 2CH₂—CH═CH—CH ₂ of DE), 1.807 and 1.783 (multiplets, 8H; 4CO—CH₂CH ₂),1.526 and 1.475 (overlapping m and t, total 193H; m, 20CH₂ of DE; t,J=7.3 Hz, 51NCH₂CH ₃), 1.063 (t, 6H, J=7 Hz; 2CH₃ of DE).

MALDI TOF mass-spectrum, M/Z: 6028, M+H; 6050, M+Na.

Preparation of3-trifluoroacetamidopropyl-3,4-di-O-acetyl-2,6-di-O-benzyl-α-D-galactopyranosyl-(1→3)-2,4-di-O-acetyl-6-O-benzyl-β-D-galactopyranosyl-(1→4)-2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-β-D-glucopyranoside(27) (Step i of Scheme VI)

The glycosyl acceptor(3-trifluoroacetamidopropyl)-2-acetamido-3-O-acetyl-6-O-benzyl-2-deoxy-4-O-(2,4-di-O-acetyl-6-O-benzyl-β-D-galactopyranosyl)-β-D-glucopyranoside(25) was prepared according to the method disclosed in the publicationof Pazynina et al (2008). A mixture of the glycosyl acceptor 25 (500 mg,0.59 mmol), thiogalactopyranoside 26 (576 mg, 1.18 mmol), NIS (267 mg,1.18 mmol), anhydrous CH₂Cl₂ (25 ml) and molecular sieves 4 Å (500 mg)was stirred at −45° C. for 30 min under an atmosphere of Ar. A solutionof TfOH (21 μl, 0.236 mmol) in anhydrous CH₂Cl₂ (0.5 ml) was then added.The reaction mixture was stirred for 2 h at −45° C. and the temperaturewas then increased to −20° C. over 4 h. The mixture was kept at −20° C.overnight. Then extra amounts of thiogalactopyranoside 26 (144 mg, 0.295mmol), NIS (66 mg, 0.295 mmol) and TfOH (5 μl, 0.06 mmol) were added andthe stirring maintained at −20° C. for 2 h before being allowed toslowly warm up to r.t. (1 h). A saturated aqueous solution of Na₂S₂O₃was then added and the mixture filtered. The filtrate was diluted withCHCl₃ (300 ml), washed with H₂O (2×100 ml), dried by filtration throughcotton wool, and concentrated. Gel filtration on LH-20 (CHCl₃-MeOH)afforded the product 27 (600 mg, 80%), as a white foam.

¹H NMR (700 MHz, CDCl₃, characteristic signals), δ, ppm: 1.78-1.82 (m,4H, CHCHC, OC(O)CH₃), 1.84-1.90 (m, 1H, CHCHC), 1.91, 1.94, 1.97, 1.98,2.06 (5 s, 5×3H, 4OC(O)CH₃, NH(O)CH₃), 3.23-3.30 (m, 1H, NCHH),3.59-3.65 (m, 1H, NCHH), 4.05 (m, 1H, H-2′), 4.33 (d, 1H, J_(1,2) 7.55,H-1^(I)), 4.40 (d, 1H, J 12.04, PhCHH), 4.42 (d, 1H, J_(1,2) 8.07,H-1^(II)), 4.45 (d, 1H, J 11.92, PhCHH), 4.48 (d, 1H, J 12.00, PhCHH),4.50 (d, 1H, J 12.00, PhCHH), 4.52 (d, 1H, J 12.04, PhCHH), 4.54 (d, 1H,J 12.00, PhCHH), 4.57 (d, 1H, J 12.00, PhCHH), 4.64 (d, 1H, J 11.92,PhCHH), 4.99 (dd≈t, 1H, J 8.24, H-2^(II)), 5.08-5.13 (m, 2H, H-31,H-3^(III)), 5.23 (d, 1H, J_(1,2) 3.31, H-1^(III)), 5.46 (d, 1H, J_(3,4)2.25, H-4^(II)), 5.54 (d, 1H, J_(3,4) 3.11, H-4^(III)), 7.20-7.40 (m,20H, ArH); 7.49-7.54 (m, 1H, NHC(O)CF₃). R_(f) 0.4 (PhCH₃—AcOEt, 1:2).

Preparation of3-aminopropyl-α-D-galactopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside(29) (Steps ii and iii of Scheme VI)

The product 27 (252 mg, 0.198 mmol) was deacetylated according toZemplen (8 h, 40° C.), neutralized with AcOH and concentrated. The TLC(CH₃Cl-MeOH, 10:1) analysis of the obtained product showed two spots:the main spot with R_(f) 0.45, and another one on the start line(ninhydrin positive spot) that was an indication of partial loss oftrifluoroacetyl. Therefore, the product was N-trifluoroacetylated bytreatment with CF₃COOMe (0.1 ml) and Et₃N (0.01 ml) in MeOH (10 ml) for1 h, concentrated and subjected to column chromatography on silica gel(CHCl₃-MeOH, 15:1) to afford the product 28 as a white foam (163 mg,77%), R_(f) 0.45 (CH₃Cl-MeOH, 10:1). The product 28 was subjected tohydrogenolysis (200 mg Pd/C, 10 ml MeOH, 2 h), filtered,N-defluoroacetylated (5% Et₃N/H₂O, 3 h) and concentrated.Cation-exchange chromatography on Dowex 50X4-400 (H⁺) (elution with 5%aqueous ammonia) gave the product 29 (90 mg, 98%) as a white foam.

¹H NMR (D₂O, characteristic signals), δ, ppm: 1.94-1.98 (m, 2H, CCH₂C),2.07 (s, 3H, NHC(O)CH₃), 3.11 (m, J 6.92, 2H, NCH₂), 4.54 and 4.56 (2d,2H, J_(1,2) 8.06, J_(1,2) 7.87, H-1^(I) and H-1^(II)), 5.16 (d, 1H,J_(1,2) 3.87, H-1^(III)). R_(f) 0.3 (EtOH-BuOH-Py-H₂O—AcOH;100:10:10:10:3).

Preparation of 3-aminopropyl2-acetamido-2-deoxy-α-D-galactopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside(33) (Steps i to iii of Scheme VII)

The glycosyl chloride3,4,6-tri-O-acetyl-2-azido-2-desoxy-β-D-galactopyranosylchloride (30)was prepared according to the method disclosed in the publication ofPaulsen et al (1978). A solution of the glycosyl acceptor 25 (420 mg,0.5 mmol), silver triflate (257 mg, 1.0 mmol), tetramethylurea (120 μl,1.0 mmol) and freshly calcinated molecular sieves 4 Å in drydichloromethane (20 ml), were stirred at room temperature in darknessfor 30 min. Another portion of sieves 4 Å was added, and a solution ofglycosyl chloride 30 (350 mg, 1.0 mmol) in dry dichloromethane (3 ml)was added. The mixture was stirred for 20 h at room temperature. Theresin was filtered and washed with methanol (4×10 ml), then solvent wasevaporated. Chromatography on silica gel (elution with 5-7% isopropanolin chloroform) yielded 407 mg (70%) of the product 31 as a mixture ofanomers (a/p=3.0 as determined by ¹H-NMR spectroscopy).

A solution of the product 31 (407 mg, 0.352 mmol) in methanol (30 ml)was subjected to hydrogenolysis over 400 mg 10% Pd/C for 16 h. Then theresin was filtered off, washed with methanol (4×10 ml) and the productconcentrated in vacuum. The dry residue was acetylated with 2:1pyridine-acetic anhydride mixture (6 ml) at 20° C. for 16 h, thereagents being co-evaporated with toluene. Two chromatography steps onsilica gel (elution with 10% isopropanol in ethyl acetate and with 5-10%methanol in chloroform) resulted in 160 mg (42%) of the product 32 and39 mg (10%) of the product 32P.

A solution of 2 M sodium methylate in methanol (200 μl) was added to asolution of the product 32 (160 mg, 0.149 mmol) in dry methanol (4 ml).The solution was evaporated after 1 h, 4 ml water added and the solutionkept for 16 h before being chromatographed on a Dowex-H⁺ column (elutionwith 1 M ammonia). The eluate was evaporated, lyophilized to yield 87.2mg (91%) of the 3-aminopropyltrisaccharide (33).

¹H NMR spectra were recorded on a Bruker BioSpin nGbH spectrometer at303K. Chemical shifts (6) for characteristic protons are provided in ppmwith the use of HOD (4.750), CHCl₃ (δ 7.270) as reference. Couplingconstants (J) are provide in Hz. The signals in ¹H NMR spectra wereassigned using a technique of spin-spin decoupling (double resonance)and 2D-¹H,¹H-COSY experiments.

The values of optical rotation were measured on a digital polarimeterPerkin Elmer 341 at 25° C.

Mass spectra were registered on a MALDI-TOF Vision-2000 spectrometerusing dihydroxybenzoic acid as a matrix.

32: ¹H-NMR (700 MHz, CDCl₃): 1.759-1.834 (m, 1H, CH sp); 1.853-1.927 (m,1H, CH sp); 1.972, 1.986, 1.996, 2.046, 2.053, 2.087, 2.106, 2.115,2.130, 2.224 (10s, 10×3H, COCH₃); 3.222-3.276 (m, 1H, NCH sp);3.544-3.583 (m, 1H, OCH sp); 3.591-3.661 (m, 2H, NCH sp, H-5a); 3.764(dd≈t, 1H, H-4a, J 8.8); 3.787 (dd, 1H, H-3b, J_(3,4) 3.7, J_(2,3) 9.9);3.836 (br. t, 1H, H-5b, J 7.3); 3.882-3.920 (m, 1H, OCH sp); 3.950 (dd,1H, H-6′c, J_(6′,6″) 10.6, J_(5,6′) 5.2); 4.009 (ddd, 1H, H-2a, J_(1,2)7.9, J_(2,3) 10.0, J_(2,NH) 9.0); 4.076-4.188 (m, 5H, H-6′a, H-6′b,H-6″b, H-5c, H-6″c); 4.415 (d, 1H, H-1a, J_(1,2) 7.9); 4.443 (d, 1H,H-1b, J_(1,2) 7.9); 4.529 (dd, 1H, H-6″a, J_(6′,6″) 12.0, J_(5,6″) 2.5);4.548 (ddd, 1H, H-2c, J_(1,2) 3.4, J_(2,3) 11.6, J_(2,NH) 9.4); 4.893(dd, 1H, H-3c, J_(3,4) 3.1, J_(2,3) 11.6); 5.021 (d, 1H, H-1c, J_(1,2)3.4); 5.039-5.075 (m, 2H, H-3a, H-2b); 5.339 (dd≈d, 1H, H-4b, J 2.9);5.359 (dd, 1H, H-4c, J_(3,4) 2.7, J_(4,5) 0.9); 5.810 (d, 1H, NHAc a,J_(2,NH) 9.0); 6.184 (d, 1H, NHAc c, J_(2,NH) 9.4); 7.310-7.413 (m, 1H,NHCOCF₃ sp). R_(f) 0.31 (EtOAc-iPrOH, 10:1). MS, m/z calculated for[C₄₃H₆₀N₃F₃O₂₅]H⁺: 1076.35, found 1076.

32β: ¹H-NMR (700 MHz, CDCl₃): 1.766-1.832 (m, 1H, CH sp); 1.850-1.908(m, 1H, CH sp); 1.923, 1.969, 1.982, 2.059, 2.071, 2.099 (2), 2.120,2.136, 2.148 (10s, 10×3H, COCH₃); 3.230-3.289 (m, 1H, NCH sp); 3.521(ddd, 1H, H-2c, J_(1,2) 8.2, J_(2,3) 11.2, J_(2,NH) 7.8); 3.548-3.591(m, 1H, OCH sp); 3.591-3.648 (m, 2H, NCH sp, H-5a); 3.743 (dd≈t, 1H,H-4a, J 8.6); 3.795 (br. t, 1H, H-5b, J 6.5); 3.852 (dd, 1H, H-3b,J_(3,4) 3.6, J_(2,3) 9.9); 3.873-3.923 (m, 2H, H-5c, OCH sp); 4.002(ddd, 1H, H-2a, J_(1,2) 8.0, J_(2,3) 9.5, J_(2,NH) 8.9); 4.039 (dd, 1H,H-6′b, J_(6′,6″) 11.6, J_(5,6′) 6.9); 4.087-4.144 (m, 3H, H-6′a, H-6″b,H-6′c); 4.160 (dd, 1H, H-6″c, J_(6′,6″) 11.2, J_(5,6″) 6.0); 4.409,4.417 (2d≈t, 2×1H, H-1a, H-1b, J 7.6); 4.519 (dd, 1H, H-6″a, J_(6′,6″)11.8, J_(5,6″) 2.5); 4.992 (d, 1H, H-1c, J_(1,2) 8.2); 5.043 (dd, 1H,H-3a, J_(3,4) 8.6, J_(2,3) 9.5); 5.066 (dd, 1H, H-2b, J_(1,2) 8.0,J_(2,3) 9.8); 5.350 (dd≈d, 1H, H-4c, J 3.2); 5.372 (dd≈d, 1H, H-4b, J3.4); 5.399 (d, 1H, NHAc c, J_(2,NH) 7.8); 5.449 (dd, 1H, H-3c, J_(3,4)3.4, J_(2,3) 11.3); 5.856 (d, 1H, NHAc a, J_(2,NH) 8.9); 7.361-7.466 (m,1H, NHCOCF₃ sp). R_(f) 0.24 (EtOAc-iPrOH, 10:1). MS, m/z calculated for[C₄₃H₆₀N₃F₃O₂₅]H⁺: 1076.35, found 1076.

33: ¹H-NMR (700 MHz, D₂O): 1.924-2.002 (m, 2H, CH₂ sp); 2.060, 2.064(2s, 2×3H, NCOCH₃); 3.102 (m≈t, 2H, NCH ₂ sp, J 6.8); 3.592-3.644 (m,1H, H-5a); 3.655 (dd, 1H, H-2b, J_(1,2) 7.9, J_(2,3) 9.9); 3.702 (br.dd, 1H, H-5b, J_(5,6′) 3.8, J_(5,6″) 8.2, J_(4,5)≤1); 3.713-3.815 (m,9H); 3.846 (dd, 1H, H-6′a, J_(6′,6″) 12.3, J_(5,6′) 5.3); 3.984-4.062(m, 4H, OCH sp, H-6″a, H-4b, H-3c); 4.123 (dd≈d, 1H, H-4c, J 2.9); 4.206(br. t, 1H, H-5c, J 6.3); 4.248 (dd, 1H, H-2c, J_(1,2) 3.6, J_(2,3)11.0); 4.542 (2d≈t, 2H, H-1a, H-1b, J 7.4); 5.100 (d, 1H, H-1c, J_(1,2)3.5). R_(f) 0.55 (MeOH-1M aq. Py.AcOH, 5:1). MS, m/z calculated for[C₂₅H₄₅N₃O₆]H⁺: 644.28; found 644. [α]_(546 nm) +128 (c 0.3; MeCN—H₂O,1:1).

33β: 1H-NMR (700 MHz, D₂O): 1.938-1.991 (m, 2H, CH₂ sp); 2.055, 2.062(2s, 2×3H, NCOCH₃); 3.100 (m≈t, 2H, NCH ₂ sp, J 6.9); 3.610 (dd, 1H,H-2b, J_(1,2) 7.9, J_(2,3) 9.9); 3.603-3.636 (m, 1H, H-5a); 3.682 (br.dd, 1H, H-5b, J_(5,6′) 4.9, J_(5,6″) 7.8, J_(4,5)≤1); 3.693-3.826 (m,11H); 3.842 (dd, 1H, H-6′a, J_(6′,6″) 12.1, J_(5,6′) 5.2); 3.934-3.972(m, 2H, H-4b, H-2c); 4.012 (dd, 1H, H-6″a, J_(6′,6″) 12.2, J_(5,6″)2.0); 4.023-4.057 (m, 1H, OCH sp); 4.175 (dd≈d, 1H, H-4c, J 2.9); 4.478(d, 1H, H-1b, J_(1,2) 7.9); 4.531 (d, 1H, H-1a, J_(1,2) 8.1); 4.638 (d,1H, H-1c, J_(1,2) 8.4). R_(f) 0.48 (MeOH-1M aq. Py.AcOH, 5:1). MS, m/zcalculated for [C₂₅H₄N₃O₁₆]H⁺: 644.28; found 644. [α]_(546 nm) +6 (c0.3; MeCN—H₂O, 1:1).

Preparation of Galili-T-17-DE (35) (Step ii of Scheme VIII)

Compound 24 (4.3 mg, 5 μmol) and Et₃N (0.5 μl) in H₂O (0.75 ml) wasadded to a stirred solution of compound 34 (5 mg, 6 μmol) in dry DMSO(0.3 mL) in 3 portions during 1.5 h. The mixture was stirred for 24 h atroom temperature and then subjected to column chromatography (SephadexLH-20, MeOH—H₂O, 3:7) to yield the crude product 35. The product waslyophilized from water, the residue was dissolved in 3 ml of water,aqueous solution of NaHCO₃ (10 mM) was added to pH 6.5 and the solutionwas lyophilized to provide 3.7 mg of the compound 35 as Na-salt.

¹H NMR (700 MHz, D₂O/CD₃OD, 2:1 (v/v), selected chemical shifts) δ, ppm:1.06 (t, J 7.03 Hz, CH₃ of DE), 1.28-1.61 (m, CH₂ of DE), 1.71-1.88 (m,—COCH₂CH ₂CH ₂CH₂CO and —COCH₂CH ₂—), 1.90-1.99 (m, OCH₂CH ₂CH₂N),2.13-2.27 (m, —CH ₂CH═CHCH ₂—, NHC(O)CH ₃), 2.35-2.58 (m, COCH₂CH ₂CH₂CH₂CO— and —COCH₂CH ₂—), 2.93-3.24 (broad.s, 8H; 4C—CH ₂NH), 4.63 (dd,J 2.49, J 12.32, C(O)OCHHCHOCH₂O—), 4.67 and 4.70 (2d, J_(1,2) 7.81,J_(1,2) 7.95, H-11, H-1^(II)), 5.30 (d, J_(1,2) 3.92, H-1^(III)),5.42-5.47 (m, —OCH₂—CHO—CH₂O—), 5.52-5.58 (m, 4H, 2×—CH═CH—). MALDI TOFmass-spectrum, M/Z: 8188 (M+Na); 8204 (M+K); 8226 (MNa+K).

Preparation of(Mal-βAla-(Gly₂CMGly)₅Gly₂-NHCH₂)₃[DE-CO(CH₂)₄CO-(Gly₂CMGly)₅Gly₂-NHCH₂]C(37) (Scheme IX)

A solution of N-maleoyl-β-alanine N′-hydroxysuccinimide ester (36) (5.3mg, 20 μmol) in MeCN (500 μL) is added in a single portion to a solutionof 25.3 mg (3.3 μmol) of compound 24 in 4 mL of 25% aqueous isopropylalcohol (IPA). The pH of the reaction mixture is adjusted to 7 to 8 withaddition of NMM (1:10 (v/v) in IPA, circa 20 μL). The clear solution iskept overnight at room temperature, and the reaction endpoint checked byqualitative spot ninhydrin test. (A negative result in the testindicates the amino component has been consumed). The solvents areremoved in vacuum using a rotary evaporator, the oily residue trituratedwith MeCN (500 μL) and the mixture sonicated for 10 minutes. The slurryobtained is transferred into an Eppendorf tube and centrifuged. Thesolid is washed repeatedly with absolute ether and MeCN (3×400 μL) withsonication followed by centrifugation until no starting reagent(Mal-βAla-ONSu) is detected by TLC (CHCl₃-MeOh—AcOH, 90:8:2 v/v). Theprecipitate after final ether wash is dried to constant weight in vacuumover 4 Å molecular sieves. A quantity of 18.9 mg (70%) of(Mal-βAla-CMG3-NHCH₂)₃CCH₂NH-CMG3-Ad-DOPE (37) was obtained as anamorphous white powder. The isolated substance may contain circa 17moles of tertiary amines and a mole of sodium ion (Na) per mole of 37.

R_(f) 0.4-0.5, (CHCl₃-MeOH—H₂O, 1:3:1 (v/v/v) plus 0.5% pyridine).

¹H NMR (700 MHz, [D₂]H₂O/[D₄]CH₃OH 1:1 (v/v), 30° C.) of Na/Et₃N salt(˜7.3 M/M Et₃N) δ, ppm: 7.038 (s, 6H; 3 CH═CH), 5.542 (m, 4H; 2 cisCH═CH of DE), 5.446 (m, 1H; OCH₂—CH(OCO)CH₂O of DE), 4.635 (dd, 1H,J=12.2 Hz/2.3 Hz; OCH₂—CH(OCO)CHOCO of DE), 4.516-4.041 (181H; 20NCH₂CO, 20NCH ₂COOH, 48COCH ₂NH, OCH ₂—CH(OCO)CHHOCO of DE, OCH ₂CH₂NH ofDE), 3.985 (t, J=6.8 Hz, 6H; 3NCH₂ of Ala), 3.594 (t, 2H, J=4.5 Hz; OCH₂CH ₂NH of DE), 3.384 (q, 44H, J=7.3 Hz; 22NCH ₂CH₃), 3.079 (broad.s,8H; 4C—CH ₂NH), 2.777 (t, 6H, J=6.8 Hz; 3CH₂CO of Ala), 2.548, 2.522,2.515 and 2.449 (triplets, total 8H; 4CO—CH ₂CH₂), 2.195 (˜dd, 8H,J=11.5 Hz/5.8 Hz; 2CH ₂—CH═CH—CH ₂ of DE), 1.812 and 1.776 (multiplets,8H; 4CO—CH₂CH ₂), 1.484 and 1.454 (overlapping t and m, total 106H; t,J=7.3 Hz, 22NCH₂CH ₃; m, 20CH₂ of DE), 1.061 (t, 6H, J=7.1 Hz; 2CH₃ ofDE).

Preparation of(MUT21-Mal-βAla-(Gly₂CMGly)₅Gly₂-NHCH₂)₃[DE-CO(CH₂)₄CO-(Gly₂CMGly)₅Gly₂-NHCH₂]C(38) (Scheme X)

A quantity (12.5 mg, 7.4 μmol) of the 14-mer oligopeptide designated

-   -   SerGlnThrAsnAspLysHisLysArgAspThrTyrProCys (SEQ ID NO: 01)        is prepared as a solution in 4 mL 0.1 M NMM in 30% aqueous        isopropyl alcohol, pH 6.6. The solution is combined with 5 mL of        the same buffer, in which a quantity (13.5 mg, 1.64 μmol) of 37        has been dissolved. The reaction mixture is stirred overnight at        room temperature and centrifuged. The supernatant is dialyzed        against unbuffered 30% (v/v) IPA-water for 24 hours and Milli-Q        water using a dialysis bag with a cutoff molecular weight of 3.5        kDa (Spectra/Por 3) to remove residual oligopeptide material.        The slurry obtained is then transferred into a lyophilization        flask and freeze-dried to a constant weight. A quantity of 18.4        mg (84%) of construct 38 is obtained as an amorphous white        powder. The expected signals ratio of low-field protons        characteristic of peptide and lipid parts of the construct is        revealed in ¹H NMR (3 mg/mL in D₂O/CD₃OD 2:1, 303 K, 700 MHz)        (FIG. 8).

Comparative Chemistry

Preparation of{[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-aceticacid methyl ester (7) (Step i of Comparative Scheme I)

An alternative method of preparing compound 7 was employed.N-Methylmorpholine (11.0 ml, 0.1 mol) was added to a stirred suspensionof Boc-glycyl-glycine (23.2 g, 0.1 mol) in 150 ml methylene chloride,the solution was cooled to −15° C. and isobutyl chloroformate (13.64 g,0.1 mol) was added for 10 min. Then 1-hydroxybenzotriazole and thesolution of (methoxycarbonylmethylamino)-acetic acid methyl ester (7)(16.1 g, 0.1 mol) in 50 ml DMF were added to the compound 39 containingreaction mixture at the same temperature. The resulting mixture wasstirred for 30 min at 0° C. then for 2 h at ambient temperature andevaporated to dryness. The residue was dissolved in 200 ml of methylenechloride and washed with 100 ml 0.5 M HCl and 200 ml 2% aq. NaHCO₃.Solvents were evaporated in vacuum and the residue was purified withcolumn chromatography on silica gel (3% MeOH in CHCl₃) to give purecompound 7 (34.08 g, 91%) as a colourless glass.

TLC: R_(f)=0.40 (5% MeOH in CHCl₃), R=0.49 (7:1 (v/v)chloroform/methanol).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.) δ, ppm: 7.826 (t, J=5.1 Hz, 1H;NHCO), 6.979 (t, J=5.9 Hz, 1H; NHCOO), 4.348 and 4.095 (s, 2H; NCH₂COO), 3.969 (d, J=5.1 Hz, 2H; COCH ₂NH), 3.689 and 3.621 (s, 3H; OCH₃), 3.559 (d, J=5.9 Hz, 2H; COCH ₂NHCOO), 1.380 (s, 9H; C(CH₃)₃).R_(f)0.49 (7:1 (v/v) chloroform/methanol).

Preparation of{[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-aceticAcid (8) (Step ii of Comparative Scheme I)

0.2 M aqueous NaOH (325 ml) was added to a stirred solution of{[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-aceticacid methyl ester (8) (24.42 g, 65.12 mmol) in methanol (325 ml),reaction mixture was kept for 15 min at ambient temperature, acidifiedwith acetic acid (5 ml) and evaporated to dryness. Column chromatographyof the residue on silica gel (methanol-ethyl acetate 1:1) gave thetarget compound as Na-salt (20.44 g) which was dissolved inmethanol/water/pyridine mixture (20:10:1, 350 ml) and passed throughion-exchange column (Dowex 50X4-400, pyridine form, 300 ml) to remove Nacations. Column was washed with the same mixture, eluate evaporated anddried in vacuum to give pure compound 8 (20.15 g, 86%) as a white solid.TLC: R_(f)=0.47 (iPrOH/ethyl acetate/water 4:3:1).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.), mixture of cis- and trans-conformersof N-carboxymethylglycine unit c.3:1. Major conformer; δ, ppm: 7.717 (t,J=5 Hz, 1H; NHCO), 7.024 (t, J=5.9 Hz, 1H; NHCOO), 4.051 (s, 2H; NCH₂COOCH₃), 3.928 (d, J=5 Hz, 2H; COCH ₂NH), 3.786 (s, 2H; NCH ₂COOH),3.616 (s, 3H; OCH ₃), 3.563 (d, J=5.9 Hz, 2H; COCH ₂NHCOO), 1.381 (s,9H; C(CH₃)₃) ppm; minor conformer, 8=7.766 (t, J=5 Hz, 1H; NHCO), 7.015(t, J=5.9 Hz, 1H; NHCOO), 4.288 (s, 2H; NCH ₂COOCH₃), 3.928 (d, J=5 Hz,2H; COCH ₂NH), 3.858 (s, 2H; NCH ₂COOH), 3.676 (s, 3H; OCH ₃), 3.563 (d,J=5.9 Hz, 2H; COCH ₂NHCOO), 1.381 (s, 9H; C(CH₃)₃). R_(f) 0.47 (4:3:1(v/v/v) i-PrOH/ethyl acetate/water).

Preparation of{[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-aceticacid N-oxysuccinimide ester (Boc-Gly₂(MCM)GlyOSu) (9) (Step iii ofComparative Scheme I)

N,N′-Dicyclohexylcarbodiimide (14.03 g, 68.10 mmol) was added to anice-cooled stirred solution of{[2-(2-tert-butoxycarbonylamino-acetylamino)-acetyl]-methoxycarbonylmethyl-amino}-aceticacid (26.40 g, 73.13 mmol) and N-hydroxysuccinimide (8.70 g, 75.65 mmol)in DMF (210 ml). The mixture was stirred for 30 min at 0° C. then for 2h at ambient temperature. Precipitated N,N′-dicyclohexylurea wasfiltered off, washed with DMF (80 ml). The filtrate and washings wereconcentrated and the residue was stirred with Et₂O (500 ml) for 1 h.Ether extract was decanted and the residue was concentrated to give

compound 9 as a white foam (32.57 g, 97%). TLC: R_(f)=0.71(acetone/acetic acid 40:1). ¹H NMR (500 MHz, DMSO[D₆], 30° C.), mixtureof cis- and trans-conformers of N-carboxymethylglycine unit c. 3:2.

Major conformer; δ, ppm: 7.896 (t, J=5.1 Hz, 1H; NHCO), 6.972 (t, J=5.9Hz, 1H; NHCOO), 4.533 (s, 2H; NCH ₂COON), 4.399 (s, 2H; NCH ₂COOCH₃),3.997 (d, J=5.1 Hz, 2H; COCH ₂NH), 3.695 (s, 3H; OCH ₃), 3.566 (d, J=5.9Hz, 2H; COCH ₂NHCOO), 1.380 (s, 9H; C(CH₃)₃).

Minor conformer; δ, ppm: 7.882 (t, J=5.1 Hz, 1H; NHCO), 6.963 (t, J=5.9Hz, 1H; NHCOO), 4.924 (s, 2H; NCH ₂COON), 4.133 (s, 2H; NCH ₂COOCH₃),4.034 (d, J=5.1 Hz, 2H; COCH ₂NH), 3.632 (s, 3H; OCH ₃), 3.572 (d, J=5.9Hz, 2H; COCH ₂NHCOO), 1.380 (s, 9H; C(CH₃)₃).

R_(f) 0.71 (40:1 (v/v) acetone/acetic acid).

Preparation of H₂N-CMG2-NH₂ (45) (Comparative Schemes II and III)

A solution of ethylenediamine (40) (808 mg, 13.47 mmol) and Et₃N (1.87ml, 13.5 mmol) in DMSO (5 ml) was added to a stirred solution ofBoc-Gly₂-(MCM)Gly-OSu (9) (15.42 g, 33.68 mmol) in DMSO (50 ml). Thereaction mixture was stirred for 30 min at ambient temperature andacidified with acetic acid (1.2 ml), then fractionated with SephadexLH-20 column (column volume 1200 ml, eluent—MeOH/water 2:1+0.2% AcOH).Fractions containing compound Boc₂MCMG (41) were combined, solventsevaporated and the residue was concentrated in vacuum. The product wasadditionally purified by silica gel column chromatography using2-propanol/ethyl acetate/water (2:6:1) as eluent. Fractions containingpure Boc₂MCMG (41) were combined, solvents evaporated and a residue wasdried in vacuum to give target Boc₂MCMG (41) as colourless foam (8.41 g,84%). TLC: R_(f)=0.48 (^(i)PrOH/ethyl acetate/water 2:3:1).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.), mixture of conformers˜3:2: 8.166,8.125, 7.917 and 7.895 (m, total 2H; 2CONHCH₂), 7.793 (m, 2H;NHCH₂CH₂NH), 7.001 (br. t, 2H; 2NHCOO), 4.277-3.893 (total 12H; 2CH₂COO, 4NCH₂CO), 3.690 and 3.635 (s, total 6H; 2COOCH₃), 3.567 (d,J=5.8 Hz, 4H; 2CH₂NHCOO), 3.131 (m, 4H; NHCH ₂CH ₂NH), 1.379 (s, 18H;2C(CH₃)₃) ppm.

MS, m/z: 769 [M+Na], 785 [M+K].

Trifluoroacetic acid (25 ml) was added to a stirred solution of Boc₂MCMG(41) (4.88 g, 6.535 mmol) in methylene chloride (25 ml) and the solutionwas kept for 1 h at ambient temperature. Then a reaction mixture wasconcentrated and the residue was evaporated three times with anhydrousMeOH (50 ml), then a residue was extracted three times with Et₂O (100ml) to remove traces of trifluoroacetic acid. The resulted precipitate(as a white solid) was dried to give 5.06 g (˜100%) of MCMG (42) asbis-trifluoroacetic salt. TLC: R_(f)=0.23 (ethanol/water/pyridine/aceticacid 5:1:1:1).

¹H NMR (500 MHz, D₂O, 30° C.), mixture of conformers˜5:4: 4.400-4.098(total 12H; 2CH₂COO, 4NCH₂CO), 3.917 (s, 4H; 2COCH₂NH₂), 3.829 and 3.781(s, total 6H; 2COOCH₃), 3.394 (m, 4H; NHCH₂CH₂NH) ppm.

MS, m/z: 547 [M+H], 569 [M+Na], 585 [M+K].

A solution of Boc-Gly₂-(MCM)Gly-OSu (9) (7.79 g, 16.994 mmol) in DMSO(17 ml) and Et₃N (2.83 ml, 20.4 mmol) was added to the stirred solutionof H₂N-MCMG-NH₂ (42) (5.06 g, 6.796 mmol) in DMSO (13 ml). The reactionmixture after stirring for 2 h at ambient temperature was acidified withacetic acid (4.0 ml) and fractionated with Sephadex LH-20 columnchromatography (column volume 1200 ml, eluent—MeOH/water 2:1+0.2% AcOH).Fractions containing pure Boc₂MCMG2 (43) were combined, solventsevaporated and the residue was dried in vacuum to give target Boc₂MCMG2(43) as colourless foam (8.14 g, 97%). TLC: R_(f)=0.25 (^(i)PrOH/ethylacetate/water 2:3:1).

¹H NMR (500 MHz, [D₆]DMSO, 30° C.), mixture of conformers: 8.393-7.887(total 6H; 6CONHCH₂), 7.775 (m, 2H; NHCH₂CH₂NH), 6.996 (br. t, 2H;2NHCOO), 4.299-3.730 (total 28H; 4CH₂COO, 10NCH₂CO), 3.691 and 3.633 (s,total 12H; 4COOCH₃), 3.564 (d, J=5.8 Hz, 4H; 2CH₂NHCOO), 3.129 (m, 4H;NHCH ₂CH ₂NH), 1.380 (s, 18H; 2C(CH₃)₃) ppm.

MS, m/z: 1256 [M+Na], 1271 [M+K].

Boc₂MCMG2 (43) (606 mg, 0.491 mmol) was dissolved in CF₃COOH (2 ml) andthe solution was kept for 30 min at r.t. Trifluoroacetic acid wasevaporated in vacuum and the residue was extracted three times with Et₂O(trituration with 25 ml of Et₂O followed by filtration) to removeresidual CF₃COOH and the obtained white powder was dried in vacuum. Thepowder was dissolved in 4 mL of water and then was freeze-dried. Yieldof H₂N-MCMG2-NH₂ (44) (TFA salt) was estimated as quantitative (actualweight was larger than theoretical by ˜10% due to stability ofhydrates). TLC: R_(f)=0.21 (ethanol/water/pyridine/acetic acid 5:1:1:1).

¹H NMR (500 MHz, [D₂]H₂O, 30° C.), mixture of conformers: 4.430-4.014(total 28H; 4CH₂COO, 10NCH₂CO), 3.911 (s, 4H; 2COCH ₂NH₂), 3.823 and3.772 (s, total 12H; 4COOCH₃), 3.386 (m, 4H; NHCH₂CH₂NH) ppm.

MS, m/z: 1034 [M+H], 1056 [M+Na].

To the solution of H₂N-MCMG2-NH₂ (44) (˜0.49 mmol) in water (20 mL) Et₃N(0.5 mL) was added, and the solution was kept for 15 h at r.t. Thereaction mixture was evaporated to dryness and the residue was desaltedon Sephadex LH-20 column (two methods): Method A. The residue wasdissolved in water (3 ml) and the solution was desalted on SephadexLH-20 column (column volume 250 mL, eluent—MeOH/water 1:1+0.05 Mpyridine acetate). Fractions, containing H₂N-CMG2-NH₂ (45) contaminatedwith salts were combined separately, evaporated and the residue wasdesalted again. Combined fractions, containing pure H₂N-CMG2-NH₂ (45),were evaporated to ˜4 ml volume and freeze dried. Yield of H₂N-CMG2-NH₂(45) (internal salt) was 431 mg (90%). Method B. The residue wasdissolved in water (3 ml) and the solution was desalted on SephadexLH-20 column (column volume 250 mL, eluent—MeOH/water 1:1+1% conc. aq.NH₃). Fractions, containing pure H₂N-CMG2-NH₂ (45), were evaporated to˜4 ml volume and freeze dried. The residue (ammonia salt of H₂N-CMG2-NH₂(45)) was dissolved in ^(i)PrOH/water 1:1 mixture (10 mL), Et₃N (0.2 mL)was added, and the solution was evaporated to dryness. This procedurewas repeated twice; the residue was dissolved in 4 mL of water andfreeze-dried. Yield of the di-Et₃N salt of H₂N-CMG2-NH₂ (45) was 549 mg(95%).

TLC: R_(f)=0.50 (^(i)PrOH/MeOH/acetonitrile/water 4:3:3:4+3% conc. aq.NH₃), or R_(f)=0.43 (^(i)PrOH/EtOH/MeOH/water 1:1:1:1, 0.75M NH₃).

¹H NMR of H₂N-CMG2-NH₂ (45) internal salt (500 MHz, [D₂]H₂O, 30° C.),mixture of conformers: 4.328-4.006 (total 28H; 4CH₂COO, 10NCH₂CO), 3.907(s, 4H; 2COCH₂NH₂), 3.381 (m, 4H; NHCH ₂CH ₂NH) ppm.

MS, m/z: 977 [M+H], 999 [M+Na], 1015 [M+K].

Preparation of H₂N-CMG2-Ad-DOPE (46) (Comparative Scheme IV)

To the intensively stirred solution of H₂N-CMG2-NH₂ (45) (425 mg, 0.435mmol of internal salt) in i-PrOH/water mixture (i-PrOH/water 3:2, 10 mL)the 1 M aq. solution of NaHCO₃ (0.435 mL, 0.435 mmol) and then thesolution of DOPE-Ad-OSu (23) (211 mg, 0.218 mmol) in dichloroethane (0.4mL) were added. The reaction mixture was stirred for 2 h and thenacidified with 0.2 mL of AcOH and evaporated to minimal volume at 35° C.The solid residue was dried in vacuum (solid foam) and then thoroughlyextracted with CHCl₃/MeOH mixture (CHCl₃/MeOH 4:1, several times with 10mL, TLC control). The extracted residue consisted of unreactedH₂N-CMG2-NH₂ (45) and salts (about 50% of H₂N-CMG2-NH₂ (45) wasrecovered by desalting of combined the residue and a fractions afterchromatography on silica gel according to procedure described in theH₂N-CMG2-NH₂ (45) synthesis). The combined CHCl₃/MeOH extracts (solutionof H₂N-CMG2-Ad-DOPE (46), DOPE-Ad-CMG2-Ad-DOPE, N-oxysuccinimide andsome H₂N-CMG2-NH₂ (45)) were evaporated in vacuum and dried. Theobtained mixture was separated on silica gel column (2.8×33 cm, ˜200 mLof silica gel in CHCl₃/MeOH 5:1). The mixture was placed on column inMeOH/CHCl₃/water mixture (MeOH/CHCl₃/water 6:3:1+0.5% of pyridine) andthe components were eluted in a stepwise ternary gradient:MeOH/CHCl₃/water composition from 6:3:1 to 6:2:1 and then to 6:2:2 (allwith 0.5% of pyridine). DOPE-Ad-CMG2-Ad-DOPE was eluted first(R_(f)=0.75, MeOH/CHCl₃/water 3:1:1), followed by desiredH₂N-CMG2-Ad-DOPE (46) (R_(f)=0.63, MeOH/CHCl₃/water 3:1:1), last elutedwas H₂N-CMG2-NH₂ (45) (R_(f)=0.31, MeOH/CHCl₃/water 3:1:1). Fractions,containing pure H₂N-CMG2-Ad-DOPE (46) were combined and evaporated todryness. To remove any low molecular weight

impurities and solubilised silica gel the residue was dissolved in^(i)PrOH/water 1:2 mixture (2 mL), and was passed through Sephadex LH-20column (column volume 130 mL, eluent—^(i)PrOH/water 1:2+0.25% ofpyridine). Fractions containing pure H₂N-CMG2-Ad-DOPE (46) were combinedand evaporated (20% of 2-propanol was added to prevent foaming) todryness, the residue was dissolved in water (˜4 mL) and freeze-dried.Yield of H₂N-CMG2-Ad-DOPE (46) was 270 mg (68% on DOPE-Ad-OSu or 34% onH₂N-CMG2-NH₂ (45)).

¹H NMR (500 MHz, [D₂]H₂O/[D₄]CH₃OH 2:1, 30° C.): 5.505 (m, 4H; 2CH₂CH═CHCH₂), 5.476 (m, 1H; OCH₂CHCH₂O), 4.626 (dd, J_(gem)=11.6 Hz, 1H;OCHCHCH₂O), 4.461-4.084 (total 37H; 4CH₂COO, 11NCH ₂CO, OCHCHCH ₂O,OCH₂CH₂N), 4.002 (s, 2H; COCH ₂NH₂), 3.573 (m, 4H; NHCH ₂CH ₂NH),2.536-2.463 (m, total 8H; 4CH₂CO), 2.197 (m, 8H; 2CH ₂CH═CHCH ₂), 1.807(m, 8H; 4CH ₂CH₂CO), 1.480 (m, 40H; 20CH₂), 1.063 (˜t, J≈6 Hz, 6H; 2CH₃)ppm.

MS, m/z: 1831 [M+H].

Preparation of Galili-CMG2-Ad-DOPE (47) (Comparative Scheme V)

To a stirred solution of compound 34 (66 mg, 0.079 mmol) in dry DMSO (6mL) were added 15 μl Et₃N and powdered H₂N-CMG2-Ad-DOPE (46) (95 mg,0.0495 mmol) in 3 portions. The mixture was stirred for 24 h at roomtemperature and then subjected to column chromatography (Sephadex LH-20,i-PrOH—H₂O, 1:2, 0.5 v % Py, 0.25 v % AcOH) to yield the crude compound47 in a form of Py-salt; The compound was lyophilized from water twotimes, then dissolved again in 10 ml of water, aqueous solution ofNaHCO₃ (50 mM) was added to pH 6.5 for obtaining the compound 47 in aform of Na-salt and the solution was subjected to lyophilization. Theyield of compound 47 (Na-salt) was 114 mg (86% based on NH₂—CMG₂-DE),R_(f) 0.6 (i-PrOH-MeOH-MeCN—H₂O, 4:3:6:4). ¹H NMR (700 MHz, D₂O—CD₃OD,1:1 (v/v), 40° C.; selected signals) δ, ppm: 1.05 (t, J 7.03 Hz, 6H;2CH₃ ), 1.40-1.58 (m, 40H; 20CH₂ ), 1.73-1.87 (m, 12H; 2×—COCH₂CH ₂CH₂CH₂CO and 2×—COCH₂CH ₂—), 1.90-1.99 (m, 2H; OCH ₂CH ₂CH₂N), 2.15-2.25(m, 11H; 2×—CH ₂CH═CHCH ₂—, NHC(O)CH ₃), 2.39-2.59 (2m, total 12H,2×—COCH₂CH ₂CH ₂CH₂CO— and 2×—COCH₂CH ₂—) 4.63 (dd, 1H, J 2.51, J 12.20,C(O)OCHHCHOCH₂O—), 4.67 and 4.69 (2d×1H, J_(1,2) 7.81, J_(1,2) 7.95,H-1^(I), H-1^(II)), 5.30 (d, 1H, J_(1,2) 3.88, H-1^(III)), 5.42-5.46 (m,1H, —OCH₂—CHO—CH₂O—), 5.49-5.59 (m, 4H, 2×—CH═CH—); MALDI TOFmass-spectrum, M/Z: 2567 (M+Na); 2583 (M+K); 2589 (MNa+Na); 2605(MNa+K); 2611 (MNa₂+Na).

Preparation of GalNAcα1-3Galβ1-4GlcNAc-Ad-DOPE (33) (Comparative SchemeVI)

To a solution of the product 23 (33 μmol) in N,N-dimethylformamide (1ml), 30 μmol of the 3-aminopropyltrisaccharide 33 and 5 μl oftriethylamine (Et₃N) were added. The mixture was stirred for 2 h at roomtemperature. Column chromatography on silica gel (CH₂Cl₂-EtOH—H₂O;6:5:1) provided an 81% yield of the construct 48.

48: ¹H NMR (700 MHz, CDCl₃—CD₃OD, 1:1 v/v, selected), δ, ppm: 1.05 (t,6H, J 7.05, 2CH₃ ), 1.39-1.55 (m, 40H, 20CH₂ ), 1.75-1.84 (m, 8H,COCH₂CH ₂CH ₂CH₂CO and 2×COCH₂CH ₂—), 1.84-1.96 (m, 2H, O—CH₂CH₂CH₂—NH), 2.15-2.22 (m, 14H, 2×(—CH ₂—CH═CH—CH ₂—), 2×NHC(O)CH ₃),2.34-2.46 (m, 4H, 2×—CH ₂—CO), 2.36-2.44 (m, 4H, 2×—CH ₂—CO), 3.29-3.34(m, 1H, —CH₂—CHH—NH), 4.17-4.20 (m, 2H, —CHO—CH ₂OP—), 4.34-4.39 (m, 2H,—CH₂OPO—CH₂ —CH₂), 4.57 (d, 1H, J_(1,2) 8.39, H-1^(I)), 4.50 (dd, 1H, J3.78, J 10.82, —C(O)OCHHCHOCH₂O—), 4.58-4.61 (m, 2H, H-1^(II),C(O)OCHHCHOCH₂O—), 5.15 (d, 1H, J_(1,2) 3.76, H-1^(III)), 5.38-5.42 (m,1H, —OCH₂—CHO—CH₂O—), 5.47-5.53 (m, 4H, 2×—CH═CH—). R_(f) 0.5(CH₂Cl₂-EtOH—H₂O; 6:5:1).

Preparation of Galα1-3Galβ1-4GlcNAc-Ad-DOPE (49) (Comparative SchemeVII)

Construct 49 was prepared according to the same method employed for thepreparation of construct 48. Eluent for column chromatography on silicagel: CH₂Cl₂-EtOH—H₂O; 6:5:1, yield of construct 49-84%;

49: ¹H NMR (700 MHz, CDCl₃—CD₃OD, 1:1 v/v, selected signals), δ, ppm:1.05 (t, 6H, J 6.98, 2CH₃ ), 1.36-1.55 (m, 40H, 20CH₂ ), 1.73-1.84 (m,8H, COCH₂CH ₂CH ₂CH₂CO and 2×(COCH₂CH ₂—), 1.85-1.96 (m, 2H, O—CH₂CH₂CH₂—NH), 2.14-2.22 (m, 11H, 2×(—CH ₂—CH═CH—CH ₂—), NHC(O)CH ₃),2.45-2.52 (m, 4H, 2×—CH ₂—CO), 2.36-2.45 (m, 4H, 2×—CH ₂—CO), 3.29-3.35(m, 1H, —CH₂—CHH—NH), 3.52-3.62 (m, 3H, PO—CH₂—CH ₂—NH, —CH₂—CHH—NH),4.13-4.18 (m, 2H, —CHO—CH ₂OP—), 4.19 (d, 1H, J_(3,4) 2.48, H-4^(II)),4.36 (dd, 1H, J 6.8, J 12.00, —C(O)OCHHCHOCH₂O—), 4.56 (d, 1H, J_(1,2)8.39, H-1^(I)), 4.60 (dd, 1H, J 2.87, J 12.00, C(O)OCHHCHOCH₂O—), 4.61(d, 1H, J_(1,2) 7.57, H-1^(II)), 5.18 (d, 1H, J_(1,2) 2.52, H-1^(III)),5.34-5.43 (m, 1H, —OCH₂—CHO—CH₂O—), 5.45-5.54 (m, 4H, 2×—CH═CH—). R_(f)0.45 (CH₂Cl₂-EtOH—H₂O; 6:5:1).

Biology

Preparation of Kodecytes

Stock solutions of constructs (35, 47, 48 and 49) were prepared at aconcentration of 1 mg/mL in a red blood cell (RBC) preservative solution(CELPRESOL™, CSL Limited). Prior to dilution each stock solution wasvortexed for 45 seconds at room temperature (r.t.). A volume of 100 μLof diluted stock solution was added to a volume of 100 μL centrifugallypacked RBCs (packed cell volume; PCV). The total volume of 200 μLsuspended RBCs was incubated at 37° C. for 2 hours before washing withCELPRESOL™ and re-suspending the modified RBCs (“kodecytes”) at aconcentration of 5% PCV in CELPRESOL™.

Preparation of Drabkins Solution

Amounts of 200 mg potassium ferricyanide (K₃Fe(CN)₆), 50 mg potassiumcyanide (KCN) and 140 mg potassium dihydrogen phosphate (KH₂PO₄) and avolume of 1 mL nonionic surfactant (Triton X-100) were dissolved indeionised water and made up to a volume of 1 L. The solution was storedin glass bottles in the dark and pH confirmed to be in the range 7.0 to7.4 before use.

Preparation of EDTA solution

Amounts of 4.45 g ethylenediaminetetraacetic acid (EDTA) as itsdipotassium salt (K₂H₂EDTA) and 0.3 g sodium hydroxide (NaOH) weredissolved in deionised water and made up to a volume of 100 mL.

Detection of Antibodies in Patient Plasma

The ability of kodecytes prepared using different constructs to detectthe presence of antibodies in samples of plasma was compared by a methodanalogous to that described in Bovin et al (2009). The results arepresented in Table 1 and are consistent with an increased avidity forMUT21 binding antibodies (if present) in the sera of subjects.

Complement Induced Cell Lysis

Prior to use kodecytes were washed and re-suspended 5% PCV in phosphatebuffered saline (PBS). Uniformity of concentration of RBCs was confirmedby adding a volume of 40 μL of kodecyte suspension to a volume of 1 mLof Drabkins solution and the absorbance measured at 540 nm againstDrabkins solution (blank). Variations in measured absorbances wasreduced to less than 10% by adjustment of suspending volume.

The ability of constructs to induce complement mediated autolysis wasevaluated by a method analogous to that described in the publication ofHenry and Komarraju (2012). For the present studies kodecytes preparedusing construct 49 were used as a 100% lysis control. A volume of 200 μLpooled AB serum and a volume of 100 μL kodecytes prepared usingconstruct 49 at a concentration of 750 μg/mL was used as the 100% lysiscontrol. A volume of 200 μL pooled AB serum and a volume of 100 μL Ogroup RBCs (prepared as kodecytes without the addition of construct) wasused as the 0% lysis control. To measure the ability of constructs toinduce complement mediated autolysis of kodecytes volumes of 200 μL ofpooled AB serum were dispensed into duplicate sets of test tubes. Avolumes of 100 μL kodecytes was added to the tubes before incubation at37° C. for 1 hour. Following incubation a volume of 1 μLethylenediaminetetraacetic acid (EDTA) as its dipotassium salt

TABLE 1 Agglutination scores determined using samples of: naturallyoccurring Mia RBCs (“positive” control), kodecytes prepared using theconstruct 38 and its monomeric counterpart at the concentrationsindicated, and unmodified RBCs (negative control). ¹the construct‘monomeric MUT21’ was prepared according to the method disclosed in thepublication of Bovin et al (2009) using construct 46. Concentration andconstruct used in the preparation of kodecytes Trimeric MUT21 (38)Monomeric MUT21¹ 0.8% 0.01 0.03 0.03 0.01 Plasma Natural mg/mL mg/mLmg/mL mg/mL 0.8% PCV sample Mia 0.00098 0.00293 0.00879 0.00293unmodified No. RBCs mM/L mM/L mM/L mM/L RBCs 3 8 0 0 0 0 0 4 10 8 10 8 30 8 8 0 0 0 0 0 9 8 0 0 0 0 0 11 10 0 0 0 0 0 12 10 0 0 0 0 0 14 8 0 0 00 0 17 10 0 0 0 0 0 18 10 0 0 0 0 0 19 10 0 0 0 0 0 20 8 0 0 0 0 0 22 100 0 0 0 0 24 10 0 0 0 0 0 25 10 0 0 0 0 0 26 8 0 0 0 0 0 27 8 0 0 0 0 029 10 0 0 0 0 0 32 10 3 5 0 0 0 33 8 0 0 0 0 0 34 8 5 8 8 0 0 35 10 0 00 0 0 36 12 8 8 3 0 0

TABLE 2 Construct used in the preparation of kodecytes and the observeddegree of cell lysis (qualitative). Construct Degree of lysis 49 Partial35 (0.66 μM) Complete 35 (0.33 μM) Complete 47 Partial 48 Complete 100%lysis control Complete 0% lysis control None

TABLE 3 Construct used in the preparation of kodecytes, absorbance (abs,540 nm) measured for duplicate samples, percentage of cells lysedrelative to 100% control and calculated percentage of cells lysed usingstandard curve. (A1 Calculated Construct Abs 1 Abs 2 and A2) Measured %% 49 .178 .187 .183 Set as 100 51 30 (0.66 μM) .351 .358 .355 194 97 30(0.33 μM) .358 .326 .342 187 93 47 .224 .243 .234 128 65 48 .349 .345.347 190 95 100% lysis control .303 .310 .307 Not applicable 85 0% lysiscontrol .027 .005 .016 Not applicable 7was added to each to each test tube to provide a final concentration of0.1 mM EDTA. The test tubes were then centrifuged and thecharacteristics of the sedimented RBCs and supernatant observed (Table 2and FIG. 2). In addition a volume of 160 μL of the cell free supernatantwas removed and added to a volume of 1 mL of Drabkins solution. Theabsorbance of the solution was then measured at 540 nm against a volumeof 160 μL pooled AB serum added to a volume of 1 mL of Drabkins solution(blank). The absorbance of the supernatant was calculated as apercentage of the initial absorbance of the suspension of kodecytes. Thepercentage of cells lysed was calculated against a standard curve.

Kodecytes prepared using the multivalent ligand construct 35 appear tobe approximately twice as sensitive to autolysis as kodecytes preparedusing the construct 49. The half molar and molar equivalents producedapproximately equal degrees of cell lysis. Kodecytes prepared using theconstruct 47 were somewhat more sensitive to lysis than kodecytesprepared using the construct designated 49. (This observation isconsistent with the observations for antibody induced agglutination withkodecytes prepared using construct 38.) Kodecytes prepared using theconstruct 48 appear to be approximately twice as sensitive to lysis askodecytes prepared using the construct 49. These observations aresubmitted to be predictive of the efficacy of the constructs whenemployed in the method of treating patients with tumours as disclosed inthe publication of Galili et al (2015).

Although the invention has been described with reference to embodimentsor examples it should be appreciated that variations and modificationsmay be made to these embodiments or examples without departing from thescope of the invention. For example, it is anticipated thatbis(N-hydroxysuccinimidyl) succinate, bis(N-hydroxysuccinimidyl)glutarate, bis(N-hydroxysuccinimidyl) pimelate andbis(N-hydroxysuccinimidyl) suberate may each be substituted for the useof bis(N-hydroxysuccinimidyl) adipate (21) in the preparation of thecompounds 23 and 34.

Where known equivalents exist to specific elements, features orintegers, such equivalents are incorporated as if specifically referredto in this specification. For example, the preparation of3-aminopropylglycosides other than those specifically described in hereare disclosed in the publications of Audibert et al (1987), Bovin et al(1993), Galanina et al (1997), Karelin et al (2010), Korchagina andBovin (1992), Korchagina et al (2009), Krylov et al (2007), Nifant'ev etal (1996), Pazynina et al (2003), Pazynina et al (2014), Ryzhov et al(2012), Sherman et al (2001), Vodovozova et al (2000) and Yashunsky etal (2016). In particular, variations and modifications to theembodiments or examples that include elements, features or integersdisclosed in and selected from the referenced publications are withinthe scope of the invention unless specifically disclaimed. It isanticipated that the 3-aminopropylglycosides disclosed elsewhere may besubstituted for the compounds 29 and 33 in the synthetic schemesdescribed here.

The advantages provided by the invention and discussed in thedescription may be provided in the alternative or in combination inthese different embodiments of the invention.

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1) A construct of the structure:

where F is an oligopeptide comprising an N-maleoyl-β-alanine conjugatedCys residue, L is a conjugated phosphatidylethanolamide and S is atetraantennary spacer of the structure:

where m is the integer 1, 2 or 3 and R is of the structure:

where M is a monovalent cation or substituent, n is the integer 2, 3, 4,5, 6 or 7, and * is the point of attachment of F or L. 2) The constructof claim 1 where M is H. 3) The construct of claim 2 where L is aconjugated phosphatidylethanolamide of the structure:

where p is the integer 3, 4 or 5, W¹ and W² are independently selectedfrom C₁₆₋₂₀-alkyl or mono- or di-unsaturated C₁₆₋₂₀-alkenyl groups and *is the point of attachment of S. 4) The construct of claim 3 where theconstruct comprises the partial structure:

5) The construct of claim 4 where the construct is of the structure:

where Xaa is an amino acid residue and i and j are independently eitherzero or integers the sum of which is in the range 5 to 30 inclusive. 6)The construct of claim 5 where i is an integer in the range 5 to 30inclusive and j is zero. 7) The construct of claim 6 where i is theinteger 13 and j is zero. 8) The construct of claim 7 where theoligopeptide is the peptide of SEQ ID NO: 01.